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ABSTRACTS
I-1
CATABOLISM OF GM2 IN MAN AND MOUSE
Yu-Teh Li and Su-Chen Li
Department of Biochemistry, Tulane
University School of Medicine, New Orleans, Louisiana 70112, USA
Tay-Sachs disease
(TSD) is caused by the deficiency of the enzyme or the activator protein
that are responsible for the catabolism of GM2. The biochemical basis
of this disease remained elusive until the discovery of two b-hexosamindase
(Hex) isozymes, Hex A and Hex B, in human spleen using p-nitrophenyl-b-GlcNAc
as substrate. This finding led to the revelation of the deficiency
of Hex A in classical TSD. In the late 1960s, based on the accumulation
of GM2 and the deficiency of Hex A in classical TSD, it was concluded that
Hex A alone was responsible for the degradation of GM2, although the Hex
A activity was not determined by using the natural substrate GM2.
By examining the degradation of GM2 in the early 1970s, we found that Hex
A required a protein cofactor, GM2 activator (GM2-Act), to assist the hydrolysis
of GM2. GM2-Act has been isolated and characterized from various
tissues and overexpressed in E. coli and other expression systems.
Although human GM2-Act was very effective in stimulating the hydrolysis
of GM2 by Hex A, it was not effective in stimulating the hydrolysis of
GA2 by the same enzyme. To explain the possible function of GM2-Act,
we have synthesized an analog of GM2, called 6'GM2 [GalNAcb1-6(Neu5Aca2-3)-Galb1-4GlcCer].
We found that the GalNAc and the Neu5Ac in 6' were readily hydrolyzed by
Hex A and sialidase, respectively, without GM2-Act. Thus, the resistance
of GM2 to enzymatic hydrolysis is due to the rigid conformation of the
terminal trisaccharide in GM2 and GM2-Act may be able to modify this conformation.
The GM2-Act isolated from mouse
shares 74.1% amino acid identity with the human GM2-Act. Between
these two activator proteins the mouse GM2-Act can efficiently stimulate
the hydrolysis of both GM2 and GA2 by Hex A and to a lesser extent also
stimulate Hex B to hydrolyze GA2. This is in contrast to the human
GM2-Act. We have subsequently identified a narrow region (Asn106
- Tyr114) in the mouse GM2-Act sequence that is responsible for stimulating
the hydrolysis of GA2. Our results provide clear evidence on the
existence of an alternative pathway for GM2 catabolism in mice by converting
GM2 to GA2 and subsequently to LacCer. These results also provide
the explanation for the lack of excessive GM2 accumulation in the Hexa
gene-disrupted mice.
I-2
GLYCOSPHINGOLIPID BIOLOGY AND DISEASE
Richard L. Proia
Genetics of Development and Disease
Branch, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, Maryland 20892 USA
Glycosphingolipids (GSL)
are universal constituents of mammalian cells whose precise functions are
only beginning to emerge. When the lysosomal degradative machinery
for GSLs is defective, severe GSL storage disorders result such as Tay-Sachs,
Sandhoff and Gaucher diseases. The neuropathologic pathways in these
disorders are poorly understood and there are no effective therapies for
the catastrophic central nervous system manifestations. We have explored
the functions of GSLs by deleting subsets of structures through disruption
of GSL-specific glycosyltransferases in mice. These GSL-deficient
mice have been instrumental in defining precise GSL functions. Gene
targeted mice have also provided valuable models of the GSL storage disorders.
Global gene analysis in these disease model mice has yielded new insights
into the neurodegenerative pathways. Finally a coordinated understanding
of GSL biology and disease have enabled novel approaches to therapy of
the GLS storage diseases
I-3
MICROGLIAL ACTIVATION AND INFLAMMATORY REACTION PRECEDING
NEURODEGENERATION IN SANDHOFF DISEASE
Ryuichi Wada1,2, Cynthia J. Tifft2,3,
and Richard L. Proia2
1Department of Pathology, Hirosaki
University School of Medicine
2Genetics of Development and Disease
Branch, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, Maryland, USA
3Department of Medical Genetics,
Children's National Medical Center, Washington, DC, USA
Sandhoff disease is
a lysosomal storage disease characterized by the absence of b-hexosaminidase
and storage of GM2 ganglioside and related glycolipids in the central nervous
system. Glycolipid storage causes severe neurodegeneration and progressive
decline of neurological function that leads to death at early stage of
life. The pathogenetic mechanisms of neurodegeneration in this disease
are poorly understood. In Sandhoff disease model mice with a disrupted
b-hexosaminidase
b-subunit
gene (Hexb), apoptotic cell death was prominent in the brainstem
and spinal cord during the rapid decline of neurological functions. To
explore the molecular machinery of neurodegeneration, cDNA microarray analysis
was done, which demonstrated an upregulation of genes related to an inflammatory
reaction and activated microglia in the spinal cord of Sandhoff disease
mice. Microglial expansion and activation were progressive and preceded
the apoptotic neuronal cell death. In a human autopsy case of Sandhoff
disease, the neuropathology was characterized by apoptotic neuronal cell
death and expansion of activated microglia with increased expression of
proinflammatory cytokine, tumor necrosis factor-a. Bone marrow transplantation
(BMT) in Sandhoff mice ameliorated neurological symptoms and prolonged
life span. BMT suppressed expansion and activation of microglia and apoptotic
neuronal cell death without a detectable decrease in GM2 ganglioside storage
in neuronal cells. This evidence suggests an inflammatory reaction mediated
by microglia is an important component of neurodegeneration in Sandhoff
disease. Thus, this lysosomal storage disease has parallels to other neurodegenerative
disorders, such as Alzheimer's and prion diseases, where inflammatory processes
are believed to participate directly in neuronal cell death.
I-4
TISSUE SPECIFIC CONTROL OF GLYCO-CHAINS
Akemi Suzuki1, Fumio Omae1, Ayako
Enomoto1, Shigemi Yoshioka1, Michiko Sekine2, Yoshiaki Kikkawa2, Choji
Taya2, Hiromichi Yonekawa3, Masaru Takenaka3, Yasuko Matsuoka3, Enyu Imai3,
Mineko Izawa4, and Reiji Kannagi4
1Sphingolipid Expression Laboratory,
RIKEN Frontier Research System, Tokorozawa, 2Department of Laboratory Animal
Science, Tokyo Metropolitan Institute of Medical Science, Tokyo, 3The First
Department of Medicine, Osaka University School of Medicine, Osaka, and
4Program of Experimental Pathology, Aichi Cancer Center, Aichi, Japan
The expression of glyco-chains is
the first step for those of glycoconjugates to carry out the physiological
functions. The expression is precisely regulated in a time and space
dependent manner. We are focusing two subjects, one is kidney tubular
cell-specific regulation of core 2 b1-6GlcNAc
transferase, and the other is brain-specific suppression of CMP-NeuAc hydroxylase.
Both cases share the phenotype that the presence of glyco-chains are supported
by the expression of enzyme activities and the enzyme activities are related
to the detectable level of their mRNAs. Elucidation of molecular
mechanisms involved in the regulation of mRNA expression is the goal of
our present research.
Gsl5 gene controls the presence
of tubular cell-specific mRNA of core 2 GlcNAc transferase. The amount
of tubular-cell specific mRNA is twenty times higher than that of ubiquitously
expressed core 2 GlcNAc transferase mRNA. DBA/2 mice have a defect
on Gsl5 gene and do not express detectable amount of tubular cell-specific
mRNA. The defect of DBA/2 mice was rescued by the transgenic
experiment with a 150 kbp long BAC clone including wild type genome of
core 2 GlcNAc transferase, indicating that the 150 kbp fragment includes
responsible element critical to kidney tubular cell-specific transcriptional
regulation.
The expression of N-glycolylneuraminic
acid (NeuGc) is controlled by the enzyme activity of CMP-NeuAc hydroxylation,
which requires three enzyme proteins, cytochrome b5, cytochrome b5 reductase,
and a terminal hydroxylase, in the presence of NADH. The terminal
hydroxylase regulates the expression of NeuGc. Mice have an intact
hydroxylase gene and various tissues of mice express the mRNA. However,
in the brain, even though it contains a very high content of NeuAc, especially
of gangliosides, mRNA is not detectable by northern blotting. Surprisingly,
the same type of regulation is conserved in human, even though human lost
the ability to produce intact hydroxylase enzyme due to the deletion of
92 bp in the genome. To elucidate the molecular mechanism involved
in the suppression, we are studying transcriptional regulation of the hydroxylase
gene.
II-1
MALIGNANT TRANSFORMATION-ASSOCIATED CHANGES IN GENE EXPRESSION OF
b-1,4-GALACTOSYLTRANSFERASES
Kiyoshi Furukawa1, Takeshi Sato1,
Shanchun Guo1,2, and Katsunori Shirane1,3
1Department of Biosignal Research,
Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan, 2Department
of Pathology, Beijing Medical University, China, 3Nissin Flour Milling
Co. Ltd., Tokyo, Japan
The b-1,4-galactosyltransferase
(b-1,4-GalT)
family consists of seven members desig-nated as b-1,4-GalTs
I-VII according to the homology distances closer from the previous enzyme
which is referred to as b-1,4-GalT
I1). Preliminary studies sug-gested that b-1,4-GalTs
I, II, III and V but not b-1,4-GalTs
IV, VI and VII are involved in N-linked oligosaccharide biosynthesis.
Since these b-1,4-GalTs
except for b-1,4-GalT
VII can galactosylate N-acetylglucosamine, whether or not novel
b-1,4-GalTs
II-VI can galactosylate N-linked oligosaccharides was investigated.
To conduct this, human b-1,4-GalT
I-VI cDNAs were independently transfected into Sf-9 cells which contain
little b-1,4-GalT
activity but contain hybrid-type
N-linked oligosaccharides mainly
terminated with N-acetylglucosamine, an acceptor-substrate for b-1,4-GalT
expressed. Lectin blot analysis of membrane glycoproteins from the
gene-transfected cells showed that several protein bands react to Ricinus
communis agglutinin (RCA)-I which interacts with oligo-saccharides terminated
with the Galb1-4GlcNAc
group. Upon treatment of blots with diplococcal b-1,4-galactosidase
or N-glycanase, no lectin-reactive bands appeared, indi-cating that
b-1,4-GalTs
I-VI expressed can galactosylate
N-linked oligosaccharides in Sf-9
cells. Upon malignant transformation of cells, amounts of highly
branched N-linked oligosaccharides increase but no significant change
in b-1,4-GalT
activity towards N-acethylglucosamine is detected. When gene
expression levels of b-1,4-GalTs
I-VI were com-pared between NIH3T3 and its transformed cell line, MTAg,
the level of b-1,4-GalT
V increased and that of b-1,4-GalT
II decreased while those of b-1,4-GalTs
I, III, IV and VI remained relatively constant2). Acceptor-specificity
analysis of b-1,4-GalTs
showed that b-1,4-GalT
V can preferentially galactosylate the GlcNAcb1-6Man
branch formed by N-acetylglucosaminyltransferase V whose increased
activity is associated with malig-nant transformation of cells. These
results indicate that the increase of b-1,4-GalT
V acti-vity with the concomitant decrease of b-1,4-GalT
II activity is important for the altered glycosylation and cellular properties
of tumor cells.
1. Furukawa, K., and Sato, T. (1999)
Biochim. Biophys. Acta 1473, 54-66.
2. Shirane, K., Sato, T., Segawa,
K., and Furukawa, K. (1999) Biochem. Biophys. Res. Commun. 265, 434-438.
II-2
CHEMICAL STRUCTURE OF THE CARBOHYDRATE MOIETY OF FUCOSE-RICH
GLYCOPEPTIDES FROM HUMAN PANCREATIC JUICE
Syuichi Yoshihara, Kazushige Ichinohe,
Shigeru Shibata, Yoshikazu Toyoki and Mutsuo Sasaki
Second Department of Surgery, Hirosaki
University School of Medicine, Hirosaki, Japan.
Human pancreatic juice,
obtained from nine patients after partial excision of he pancreas for bile
duct cancer, was fractionated in order to isolate its glycopeptides. Three
glycopeptides (GP-2,GP-3 and GP-4) were purified employing ion-exchange
chromatography and gel-filtration.
All the glycopeptides
were found to be free of sialic acid and galactosamine but to have an unusually
high content of l-fucose. The chemical structures of the three glycopeptides
were determined using 500-MHz [1H]-NMR spectroscopy. One of them, glycopeptide,
GP-4, possessed a biantennary structure with three l-fucose residues. The
second glycopeptide, GP-3, had a triantennary structure with four l-fucose
residues, and the third one, GP-2, had a tetraantennary structure with
five l-fucose residues.
The chemical compositions
of these glycopeptides, including the absence of sialic acid and the high
l-fucose content, indicate that they represent a new class of glycopeptide
present in the normal human pancreas. We are investigating the relationship
to pancreas cancer and pancreatic stones.
References
1. Yoshihara S, Matsue H, Sasaki
M, Shibata S, Konn M, Fukuzawa A, and Endo M
(1995) Int. J. Pancreatol., 17,181-187
2. Yoshihara S, Sasaki M, Kawasaki
H, Yokoyama M, Endo M, and Konn M
(1993) Int. J. Pancreatol., 14,
219-225
III-1
MUC1 AS A HUMAN TUMOR MARKER
Kohzoh Imai, Toshiaki Hayashi, Takamaro
Suwa, Yusuke Makiguchi, Fumio Itoh,
Yuji Hinoda, and Tohru Takahashi
First Department of Internal Medicine,
Sapporo Medical University School of Medicine, Sapporo, Japan
MUC1 is a highly immunogenic
epithelial mucin that serves as a tumor-associated antigen in breast, pancreatic
and ovarian carcinomas. We reported, for the first time, the expression
of MUC1 on myeloma cells and the establishment of an HLA-unrestricted cytotoxic
T lymphocytes (CTL) line TN that recognized MUC1 from peripheral blood
mononuclear cells in a multiple myeloma patient. In this study, we
attempted to induce such CTL from six other multiple myeloma patients consecutively.
Bone marrow mononuclear cells were used to induce CTL, because they contain
myeloma cells that might stimulate the autologous lymphocytes. The
CTL line TS was CD8+ cell dominant and KY was CD4+ cell dominant.
Both CTL lines lysed MUC1 + myeloma and breast carcinoma cell lines.
The cytotoxicity of the CTL lines was inhibited by anti-CD3, anti-ab TCR
and anti-MUC1 mAb. The reactivity of anti-MUC1 core protein mAb and
the cytotoxicity of the CTL against the MUC1 transfectant was enhanced
by the treatment of the cells with an O-glycosylation inhibitor.
Thus it is generally accepted that the HLA-unrestricted CTL which directly
recognize the underglycosylated form of MUC1 using their TCR could be induced
from a certain proportion (about 30%) of untreated multiple myeloma patients.
On the other hand, MUC1
mucin is an anti-adhesion molecule expressed in a wide variety of tumors．To
examine whether MUC1 mucin is involved in tumor invasion, we have prepared
MUC1 transfectants and performed an in vivo tumor assay by transplanting
these into nude mice. Tumor weight after s.c. injection of MUC1 transfectants
was increased compared to that of mock transfectants. Furthermore,
MUC1-transfectant tumors invaded into the muscle layer. In vitro
invasion, adhesion to extracellular matrix components and phagokinetic
track motility were then evaluated to analyze the mechanisms for the in
vivo invasiveness of the transfectants. MUC1 transfectants exhibited an
increased in vitro invasiveness, and increased motility．These effects
of MUC1 mucin over-expression in tumor cells were abolished by the treatment
of transfectants with an inhibitor of O-glycan biosynthesis，benzyl-a-GalNAc．Our
data suggest that MUC1 mucin could be related to the increased invasive
ability of tumor
cells, whereas O-glycan might play
an essential role.
III-2
CHARACTERIZATION OF MUCIN IN WHOLE-GUT LAVAGE FLUID OBTAINED FROM
PATIENTS WITH INFLAMMATORY BOWEL DISEASE
Hiromi Saitoh, Haruhiko Tanaka, Kazunori
Muramoto, Seiji Kimura, Koumei Kubo, Masaharu Kasai, Yutaka Yoshida, Akihiro
Munakata
First Department of Internal Medicine,
Hirosaki University School of Medicine, Hirosaki, Japan
Interest in mucin has increased
considerably in recent years because of its possible etiopathogenic relationship
with ulcerative colitis (UC) and Crohnﾕs disease (CD). However it
is very difficult to separate abundant mucin which was extract from tissue
specimen. We collect mucin secreted into the gastrointestinal lumen
by simple method. Using this sample, the biochemical features of
the mucin from inflammatory bowel disease were investigated.
The control subjects
were five healthy volunteers. Four of the patient had UC and four
of the patients had CD. All subjects drank the polyethylene glycol-based
electrolyte lavage solution for bowel preparation prior to colonoscopy.
Five hundred mililiters of effluent of whole gut lavage fluid was collected
from each subject. The mucin was separated into four fractions by
Sepharose CL-4B, Sepharose CL-2B, and DEAE Sephacel chromatography.
Compared with healthy
subjects, the total yields of mucin from UC were low due to a deficiency
of neutral mucin, whereas those from CD were high, which was attributable
mainly to high-molecular-weight mucin. The fucose and sulfate contents
were low in UC, but only the former was low in CD1).
This study is the first
to demonstrate that harvesting of whole gut lavage fluid is a simple method
for collecting mucin secreted into the gastrointestinal lumen. Furthermore,
the results show that differences in the quantity and biochemical features
of mucin in the whole gut lavage fluid obtained from patients with inflammatory
bowel disease may reflect the mucosal changes associated with UC and CD.
1. Hiromi Saitoh., Keiichi Takagaki.,
Toshiya Nakamura., Akihiro Munakata., Yutaka Yosida., and Masahiko Endo;
(1996) Digestive Disease and Sciences 41.,1768-1774.
IV-1
THE RATIONALE FOR AND RESULTS OF PRE-CLINICAL TRIALS OF
CHONDROITINASE ABC IN CHEMONUCLEOLYSIS
Mark D. Brown
Department of Orthopedics and Rehabilitation,
University of Miami School of Medicine, Miami, FL, USA
Chemonucleolysis (dissolution
of the nucleus pulpous of the intervertebral disc) with chymopapain, a
proteolytic enzyme from papaya fruit, has been utilized to treat symptomatic
spinal disc herniation in humans and dogs since 1959. Pre-clinical
and subsequent human clinical trials with chymopapain showed that this
enzyme is allergenic, neurotoxic, and causes more pain following treatment
than does surgical disc excision. Allergenicity is felt to be secondary
to the high prevalence of pre-sensitization to papaya enzymes in humans.
Neurotoxicity is secondary to the non-specific proteolytic action of the
enzyme with its damaging effects on microvasculature. The increased
pain response in the immediate post-injection period is probably related
to pain mediators released from the nucleus pulposus of the disc at the
time of chemonuclelysis. An alternative enzyme that does not have the deleterious
effects of chymopapain is felt to be desirable.
The rationale for the use of
chondroitinase ABC (CABC) in chemonucleolysis are threefold. It is
anticipated that there will be a very low prevalence of pre-sensitization
to this enzyme in the human population. The mode of action of chondroitinase
ABC is highly specific to the disaccharide polymer side chains of the proteoglycan
molecule in the nucleus pulposus. Pre-clinical trials demonstrate
no deleterious effect of CABC on capillaries of the hamster cheek pouch
or the pia-arachnoid membrane of the cauda equina in the subarachnoid space
of rabbits
Injection of chondroitinase
ABC into normal intervertebral discs of rabbits, pigs, rhesus monkeys,
baboons, and goats induces disc narrowing, water loss, and depletion of
polysaccharides. Compared to chymopapain, chondroitinase ABC is not
as drastic a chemonucleolytic agent. Restoration of nucleus pulposus
matrix and subsequent disc height occurs more frequently following injection
of chondroitinase ABC compared to chymopapain. Chondroitinase ABC
was found to be effective in removing autologous nucleus pulposus from
the epidural space in rabbits. In addition, chondroitinase ABC was
effective in chemonucleolysis of a degenerative disc animal model.
Finally, chondroitinase ABC has been utilized therapeutically in dogs suffering
from herniated discs with favorable clinical results.
The pre-clinical trials of
chondroitinase ABC in chemonucleolysis have substantiated the theoretical
advantage of this enzyme over chymopapain in chemonucleolysis. The
highly specific action of CABC to polysaccharides found in the nucleus
pulpusus of the intervertebral disc and the benign action of this enzyme
on surrounding vascular and neurological tissues make it an ideal enzyme
for chemonucleolysis in humans. Whether chondroitinase ABC will initiate
less pain response in the post-injection period than chymopapain or collagenase
is a question that remains to be answered in carefully performed clinical
trials. The answer to this question will help us to understand the
basic mechanisms for pain induction from breakdown products of nucleus
pulposus.
IV-2
CHANGES IN GLYCOSAMINOGLYCAN CHAINS OF NUCLEUS PULPOSUS PROTEOGLYCAN
OF BABOON VERTEBRAL DISCS WITH AGING
Wataru Nakamura, Masahiro Yukawa,
Kazunari Takeuchi, Seiko Harata
Department of Orthopaedic Surgery,
Hirosaki University School of Medicine, Hirosaki, Japan
Age-related changes in the
glycosaminoglycans of the large proteoglycan in the nucleus pulposus of
baboon intervertebral discs were examined. From specimens of nucleus pulposus
divided into three groups according to age, the proteoglycans were separated
by column chromatographies. After enzymatic digestion,
the glycosaminoglycan chains obtained from the
proteoglycan, and analyzed using HPLC and a biosensor. The glycosaminoglycans
were composed mainly chondroitin 6-sulfate. The molecular mass of the glycosaminoglycans
decreased with aging, but their sulfate content did not change. The affinities
of the glycosaminoglycans for hydroxyapatite and for various types
of collagen were determined. Binding of glycosaminoglycans to hydroxyapatite
showed no change with aging, whereas there was increased binding to type
IX collagen with aging. Type IX collagen to bind may be influence the character
and the structure of extra-cellular matrix of vertebral discs by changing
of interruption between the glycosaminoglycan and type IX collagen.
IV-3
FEMALE INFERTILITY DUE TO CUMULUS MASS DEFECT IN BIKUNIN-DEFICIENT
MICE:
ROLES OF SHAP-HYALURONAN COMPLEX
Zhuo L1, Yoneda M1,
Zhao M1, Yingsung W1, Yoshida N2, Kitagawa
Y2, Kawamura K3, Suzuki T3, and Kimata
K1
1Institute for Molecular
Science of Medicine, Aichi Medical University, Aichi, 2Graduate
Program for Regulation of
Biological Signals, Graduate
School of Bioagriculture Sciences, Nagoya University, Nagoya, 3Japan
SLC Inc., Hamamatsu,
Japan
SHAPs (Serum-derived
Hyaluronan-Associated Proteins), the heavy chains of plasma iner-alpha-trypsin
inhibitor (IalphaI) family, are so far only the proteins that have been
shown to be covalently bound to hyaluronan (HA). The complex has been named
SHAP-HA complex and the formation was found to require the intact IalphaI
family molecules and serum protein factor(s) in addition to HA. The complex
is found in any tissues and cells where HA and serum meet. However, biological
significances of the SHAP-HA complex remain to be studied. Bikunin, the
light chain of IalphaI family, is essential for the assembly of IalphaI
family molecules and thus for formation of the SHAP-HA complex. In present
study, bikunin-defective mice were created to evaluate functions as well
as formation mechanisms of the SHAP-HA complex. Bik-/- mice were able to
grow up to adult without apparent abnormalities but showed a severe infertility
only in female mice. Histology of ovaries in Bik-/- female revealed the
normal follicle development at various stages. However, the cumulus expansion
was impared in preovulatory follicles. Oocytes in oviducts of Bik-/- females
at 0.5 dpc were greatly decreased in number (56.8%) and, more stringly,
were completely free of cumulus masses. Such naked oocytes remained uncleaved
at 1.5 dpc, indicating a unfertilized states. Together with the molecular
evidences, we conclude that the defect in the cumulus matrix was due to
the inability of the SHAP-HA complex formation and led to the female infertility.
The SHAP-HA complex therefore plays a critical role in maintenance of the
expanded cumulus mass. The mechanism may be operative in some female infertility
in human and possible therapeutic methods have been tried for rescueing.
V-1
USE OF GLYCOSAMINOGLYCANS FOR THE TREATMENT OF INTERSTITIAL CYSTITIS -
A
STRATEGY TO IMPROVE EFFICACY
V.P.Bhavanandan1, D.R. Erickson2,
N. Herb1, M. Sheykhnazari1, and S. Ordille2.
1Department of Biochemistry and Molecular
Biology, 2Department of Surgery,
The Pennsylvania State University
College of Medicine, Hershey, PA, USA.
Interstitial cystitis is a chronic
and incurable disease with symptoms including urinary urgency, frequency,
nocturia and bladder pain. A recent survey estimated between 130,000
to 170,000 cases of the disease in the United States. The most popular
etiologic theory about interstitial cystitis is that the glycocalyx of
the bladder epithelium is deficient. Accordingly, current treatments
are aimed at replacing the missing glycoconjugates by intravesical administration
of heparin, hyaluronic acid or pentosan polysulfate (Elmiron). It
is presumed that these anionic polysaccharides act by replacing the missing
coat of endogenous glycosaminoglycans on the epithelium. However,
we found that while the bladder epithelial surface contained substantial
quantities of mucin glycoproteins, such as MUC1, the levels of glycosaminoglycans
were insignificant (Buckley et. al.1) ).
We are investigating the binding
and retention of intravesically administered glycoconjugates on the bladder
surface. Biotinylated pentosan sulfate, heparin, hyaluronic acid,
and various native and asialo mucins were prepared and tested for binding
to paraffin sections of rabbit and human bladder. The binding of
radiolabeled heparin and pentosan sulfate to fresh rabbit bladders was
also tested. The mucins showed strong staining of the epithelium
indicative of lectin mediated interactions. In contrast, the anionic
polysaccharides showed either no or very weak binding in agreement with
the lack of attachment sites for these negatively charged molecules.
In other studies, we are developing
methods to improve the attachment of sulfated polysaccharides to the bladder
epithelium. The strategy is to modify the polysaccharides with specific
saccharide ligands, so that they will bind to the endogenous lectins in
the bladder. The results of histochemical studies on rabbit and human
bladder suggested the presence of galactose- and N-acetylglucosamine-binding
lectins in the epithelium (Puch and Bhavanandan2)). The presence
of galectin-3 and galectin-4 in bladder mucosa was confirmed by molecular
biological techniques. Lactose, N-acetyllactosamine and other ligands
of galectins are being used to modify biotinylated heparin and pentosan
sulfate to improve their binding to the bladder surface. [Supported
by the U.S. Public Health Service grant DK 57266]
1. Buckley, M., Xin, P., Washington,
S., Herb, N., Erickson, D.R., and Bhavanandan, V.P. (2000)
Arch. Biochem. Biophys. 375, 270-277.
2. Puch, S., and Bhavanandan, V.P.,
(1999) Urology 53, 848-852.
V-2
ENZYMATIC RECONSTRUCTION OF GLYCOSAMINOGLYCAN OLIGOSACCHARIDES
Keiichi Takagaki, Ikuko Kakizaki,
Mito Iwafune, Keinosuke Ishido and Masahiko Endo
Department of Biochemistry, Hirosaki
University School of Medicine, Hirosaki, Japan
Glycosaminoglycans
(GAGs) have many kinds of structural domains, which are known to participate
in specific physiological functions such as anticoagulant or antithrombotic
activity. However, the relationship between their biological functions
and structure is not yet fully understood. In particular, the biological
implications of the position of the sulfate as well as intrachain variation
in sulfate position are unknown. It is therefore important to develop
a method of reconstructing GAG oligosaccharides in order to investigate
their structure-function relationships.
It is known that many
glycosidases catalyze a transglycosylation reaction as a reverse reaction
in addition to their main hydrolysis reaction. Therefore, the transglycosylation
mechanism of testicular hyaluronidase, which is an endo-b-N-acetyl-hexosaminidase,
was investigated with the aim of performing enzymatic synthesis of GAG
sugar chains. It was found that disaccharide units (glucuronic acid b1-3-N-acetylglucosamine)
are successively released from the nonreducing terminal of a donor hyaluronic
acid (HA) and rapidly transferred to the glucuronic acid residue at the
nonreducing terminal of an acceptor HA via a b1-4
linkage1). Furthermore, the efficiency of the transglycosylation reaction
increases when the pH and NaCl concentration are optimized. By repeating
the transglycosylation using suitable combinations of hyaluronic acid (HA),
chondroitin (Ch), chondroitin 4-sulfate (Ch4S), chondroitin 6-sulfate (Ch6S),
chondroitin sulfate D (ChS-D), chondroitin sulfate E (ChS-E), and desulfated
dermatan sulfate (desulfated DS) as acceptors and donors, it was possible
to custom-synthesize GAG oligosaccharides2-4). As a result, GAG oligosaccharide
library, which has a hybrid structure composed of disaccharide units derived
from Ch, Ch4S, Ch6S, ChS-D, ChS-E, and desulfated DS was prepared. The
custom-synthesized GAG oligosaccharides promise to open a new avenue in
Glycomedicine.
1. Takagaki, K., et al., (1994)
Biochemistry, 33, 6503-6507.
2. Takagaki, K., et al., (1999)
Biochem. Biophys. Res. Commun., 258, 741-744.
3. Takagaki, K., et al., (2000)
J. Biochem. , 127, 695-702.
4. Takagaki, K., et al., (2000)
Biochem. Biophys. Res. Commun., 270, 588-593.
V-3
4-METHYLUMBELLIFERONE INHIBITS HYALUYRONATE SHYNTHESIS IN CULTURED
FIBROBLASTS FROM THE HUMAN UTERINE CERVIX
Kanji Tanaka, Tsuyoshi Higuchi, Shuhei
Sato, and Yoshiharu Saito.
Department of Obstetrics and Gynecology,
Hirosaki University School of Medicine
Hyaluronate (HA) is
one of the primary constituents of the extracellular matrix of the human
uterine cervix. It has been reported that during cervical ripening the
amount of HA increase to about 10 times the non-pregnant level, and decreases
rapidly back to the non-pregnant level after parturition. These dramatic
changes suggest that HA plays an important role in the regulation of cervical
function during parturition.
In a previous study,
we found that progesterone induces the rapid conversion of HA metabolism
from the synthesis phase to the degradation phase in these cultured cells.1,2
These results suggested that progesterone avoids preterm delivery by suppressing
HA synthesis. On the other hand, we reported that 4-methylumbelliferone
(MU) suppresses HA synthesis by cultured human skin fibroblasts.3
In the present study,
the effects of MU on the synthesis of HA were investigated in cultured
fibroblasts from the human uterine cervix. The cells were incubated with
[3H]glucosamine in the presence of various concentrations of MU and the
effects of MU on HA synthesis were investigated. The cells were preincubated
in media that contained various concentrations of MU. After 72 h, the medium
was removed and the cells were incubated with fresh
medium containing [3H]glucosamine without MU for 48 h. The
amount of [3H]HA synthesized was measured.
In these cells, HA
synthesis was suppressed in a dose-dependent manner. After MU was removed
from culture medium, HA synthesis started again and the amount of [3H]HA
synthesized was restored to the control level. It follows that inhibition
of HA synthesis by MU was reversible in human uterine cervix.
The MU-induced suppression
of HA synthesis may contribute to the control of cervical ripening during
human pregnancy. Further study of the mechanisms of the effects of MU on
both HA and other constituents of the extracellular matrix are required.
1. Tanaka K, Nakamura T, Higuchi
T, Saito Y, Takagaki K, Endo M, (1994) FEBS Lett, 347, 95-98.
2. Tanaka K, Nakamura T, Takagaki K, Funahashi M, Saito Y, Endo M. (1997)
FEBS Lett, 402, 223-226. 3. Nakamura T, Takagaki K, Shibata
S, Tanaka K, Higuchi T, Endo M. (1995 ) Biochem. Biophys. Res.
Commun., 208, 470-475.
P-1
ORGANIZATION AND TRANSCRIPTIONAL CONTROL OF HUMAN PLASMA MEMBRANE
SIALIDASE GENE
Kazunori Yamaguchi, Yukiko Shimada,
Tadashi Wada and Taeko Miyagi
Division of Biochemistry, Research
Institute, Miyagi Prefectural Cancer Center, Natori, Japan
Sialidase reaction is an initial
step of the degradation of glycoproteins and gangliosides. Sialidases of
mammalian origin have been implicated not only in lysosomal catabolism
but also in modulation of functional molecules involved in many biological
processes. However, the physiological significance and the regulation mechanism
of desialylation remain obscure because the structure and function of mammalian
sialidase genes are not fully understood.
We previously isolated a cDNA clone
encoding human plasma membrane sialidase (hmSD) that is specific for ganglioside1)
and therefore probably plays as a modulator of gangliosides at cell surface.
Northern blot analysis showed relatively high expression of hmSD gene in
skeletal muscle, heart and testis and very low expression in digestive
organs. An elevated expression of this gene was observed by RT-PCR studies
in various cancers including colon and gastric cancers.
To understand the regulation mechanism
of the expression of hmSD gene, we isolated the genomic clones using the
cDNA for hmSD as a probe and analyzed the genomic structure. The cosmid
and phage genomic clones isolated cover the 5ﾕ portion of the gene and
contain four exons, two of which encode open reading frame.
To characterize the regions participating
in regulation of the expression, we constructed chimeric reporter vectors
in which various lengths of 5ﾕ-franking region of the hmSD gene were fused
to promoterless luciferase gene. Transient expression of these reporter
constructs in several human cell lines indicated that 5ﾕ-franking region
of 600bp, which lacks canonical TATA box, has promoter activity in the
cell lines tested.
1. Wada, T., Yoshikawa, Y., Tokuyama,
S., Kuwabara, M., Akita, H. and Miyagi, T. (1999) Biochem. Biophys. Res.
Commun., 261, 21-27
P-2
SYNTHETIC STUDY OF NEW SIALIC ACID CONTAINING POLYMERS AS INHIBITORS OF
HEMAGGLUTINATION BY INFLUENZA VIRUSES
Yutaka Makimura1, 2, Gan Zhonghong2,
and Renｽヽoy2
1Division of Life Science, Graduate
School of Biostudies, Kyoto University, Kyoto, Japan, and 2Department of
Chemistry, University of Ottawa, Canada
Influenza viruses are one of the
most serious pathogens for human. They are known to infect host tissues
by first binding to sialoconjugates on cell surface through hemagglutinin.
a-Sialoside
containing polymers have been demonstrated for potent inhibitors of the
hemagglutination on human erythrocytes by influenza viruses. Moreover,
They have been turned out more superior antigenic properties to their corresponding
sialoconjugates from the observations of multivalent and cluster effect.
For the viewpoint of drug targeting,
we have been tried to develop efficient synthesis of sialoconjugate polymers.
We report the novel synthesis of sialocopolymers using Curtius rearrangement
reaction1).
The condensation of branched a-sialoside,
prepared from p- nitrophenyl sialic acid and 3.3-iminobis(propylamine),
with 1,4-diisocyanatebutane and 1,6-diisocyanatehexane afforded copolymers
without any promoters.
Those copolymers retained their
specific lectin binding properties as determined with wheat germ agglutinin
(WGA), a plant lectin known to bind sialosides and glucosaminides in the
agar gel double immunodifusion test. Each copolymer was observed
clearly binding. Turbidimetric assays were also tested. They
exhibited strongly cross-linking properties with WGA and copolymer including
hexyl group showed little stronger binding properties than butyl one.
These copolymers would be useful
for new potential inhibitors to the hemagglutinination of human erythrocytes
by influenza virus.
1. K. Ninomiya, T. Shioiri, S. Yamada,
Tetrahedron, 1974, 30, 2151; Idem, Chem. Pharm.bull., 1974, 22, 1398.
P-3
STUDY OF AUTOANTIBODY AGAINST ADVANCED GLYCATION ENDPRODUCTS OF THE
MAILLARD REACTION
Norie Araki1, Rie Shibayama3, Ryoji
Nagai2, Tomohiro Araki4, and Seikoh Horiuchi2
1Department of Tumor Genetics and
Biology, 2Department of Biochemistry, Kumamoto
University School of Medicine, Kumamoto,
3Pharmaceutical Research Center, Nisshin Flour Milling Co., Ltd., and 4Department
of Bioscience, Kyushu Tokai University School of Agriculture, Kumamoto,
Japan
Prolonged incubation
of proteins with reducing sugar proceeds, through formation of early-stage
products such as Schiff base and Amadori products, to the advanced glycation
end products (AGE). AGE are characterized by fluorescence, brown color
and cross-linking and implicated as factors for aging and age-enhanced
disease states such as diabetic complications. We previously demonstrated
the presence of several AGE structures in human and animal tissues using
anti-AGE antibodies1)2). Recently, we found that Ne-(carboxymethyl) lysine
(CML) is an epitope structure of the monoclonal anti-AGE antibody (6D12)3).
These findings suggest that CML as well as other AGE structures present
in vivo could serve as immunogens to generate autoantibodies. This suggestion
was tested in the present study using plasma and tissue samples from rat
and humans4).
First, plasma
samples from diabetic rats reacted positively with AGE bovine serum albumin
(BSA). These reactivities increased with the duration of diabetic states
and were inhibited specifically by CML-BSA. Second, a fraction purified
from plasma of diabetic patients, which bound to AGE-BSA, showed a positive
reaction to CML-BSA and furthermore also to human lens proteins, which
are known to undergo CML modification in vivo. Finally, patients with renal
failure caused by diabetes or nondiabetic pathologies had a higher autoantibody
activity against CML structure than that in normal subjects or diabetic
patients without renal failure. These results indicate that CML accumulated
in vivo serves as an immunological epitope to generate an autoantibody
specific for CML that might be used as a potential marker for diabetic
nephropathy or chronic renal failure.
1) Araki, N., et. al. J.Biol.Chem.
267: 10211(1992). 2) Horiuchi,S., Araki,N., et al J.Biol.Chem. 266:7329(1991)
3) Ikeda,,K., et al. Biochemistry 35:8075(1996)
4) Shibayama, R., Araki, N.,
et. al. Diabetes. 48:1842(1999)
P-4
ANALYSIS OF CARBOXYMETHYL AND CARBOXYETHYL-LYSINE IN AGE-PROTEIN OF THE
MAILLARD REACTION
Tomohiro Araki1, Ryoji Nagai2, Norie
Araki3, Seikoh Horiuchi2
1Department of Bioscience, Kyushu
Tokai University School of Agriculture, Kumamoto, 2Department of Biochemistry,
Kumamoto University School of Medicine, Kumamoto, and 3Department of Tumor
Genetics and Biology, Kumamoto University School of Medicine, Kumamoto,
Japan
Long-term incubation of protein with
glucose leads through Schiff base and Amadori product, to the formation
of advanced glycation end product (AGE) of the Maillard reaction. Immunohistochemical
study using anti-AGE antibody have detected AGE modifications in several
pathological tissues such as diabetic nephropathy. Recent study demonstrated
Ne-(carboxymethyl)lysine (CML) in several tissue proteins. Further, Ne-(carboxyethyl)lysine
(CEL) is found as methylglyoxal-derived AGE structure.
To elucidate the mechanism of the
formation of AGE structure during incubation of protein with glucose, we
analyzed CML and CEL in AGE structure by the detection methods of immunochemical
procedure using monoclonal antibody against CML and CEL, and Hitachi Model
L-8500A amino acid analyzer with cation exchange-ninhydrin system. In this
study we found that CML is a major immunological epitope in proteins modified
with AGE, and the formation of CML from Amadori product was mediated by
hydroxy radical generated by the reaction of Fe2+ with H2O2. Further, the
conversion of CML from Amadori product was enhanced in alkaline condition.
For understanding the pathway of CEL formation, we analyzed the effect
of aminoguanidine and phosphate on the formation of CEL. The result indicated
that the CEL formation in glucoase-modified proteins was inhibited by aminoguanidine
but enhanced by phosphate. From this result, we conclude that methylglyoxal
generated during incubation of protein with glucose is responsible for
protein modification by CEL. Further, the innmunohistochemical analysis
using CEL-specific monoclonal antibody demonstrated the presence of CEL-modified
proteins in various human tissues, preferentially inside the cells.
1. Ikeda, K., Higashi, T., Sano,
H., Jinnouchi, Y., Yoshida, M., Araki, T., Ueda, S., and Horiuchi, S. (1996)
Biochemistry, 35, 8075-8083
2. Nagai, R., Ikeda, K., Higashi,
T., Sano, H., Jinnouchi, Y., Araki, T., and Horiuchi, S. (1997) Biochem.
Biophys. Res. Commun., 234, 167-172
3. Nagai, R., Ikeda, K., Kawasaki,
Y., Sano, H., Yoshida, M., Araki, T., Ueda, S., and Horiuchi, S. (1998)
FEBS Lett., 425, 355-360
P-5
OCCURRENCE OF SHORTER CHAIN (C55-C60) POLYPRENOL IN YEAST
Seiji Tateyama and Hiroshi Sagami
Institute for Chemical Reaction Science,
Tohoku University, Sendai, Japan
Yeast #64 mutant was
isolated as a temperature-sensitive mutant that exhibits defects in protein
glycosylation with a reduced pool of endogenous dolichyl phosphate1). In
the present study, we tried to perform complementation analysis, with a
special attention to the endogenous polyisoprenoids contents.
Transformants were
selected for growth at 36�C, and three transformants were screened. Three
kinds of plasmids isolated from the transformants (#1, #2, and #3) were
analyzed to possess a fragment of chromosome IV, VII, and XIII, respectively.
Endogenous dolichyl-P content of #64 mutant, #1, #2, #3, and the wild type
were 2.2, 3.4, 3.0, 1.8, and 10.0 ｵg/1010 cells, respectively.
In the case for endogenous dolichol, the content were 3.8, 4.0, 0.8, 3.2,
and 2.4 ｵg/1010 cells, respectively. No polyprenol with the
same chain length as that of dolichol (C75-C80) was detected in these five
kinds of yeast cells. Surprisingly, shorter-chain (C55-C60) polyprenol
was detected in #64 mutant (1.0 ｵg/1010 cells), #1 (1.5 ｵg/1010
cells), and #3 (1.9 ｵg/1010 cells), but not in #2 and the wild
type.
These results indicate
that #64 is rescued with the increased pool of endogenous dolichyl-P (#1
and #3) or with disappearance of shorter-chain polyprenol (#2). The latter
rescue (#2) might be explained as follows. Since the dolichyl-P content
of #64 and #2 is almost same, shorter-chain polyprenol-derived compounds
(putative shorter-chain dolichyl-P) act as sugar carrier-lipids like long-chain
dolichyl-P. It is very interesting to assume occurrence of shorter-chain
sugar carrier-lipids to see the biosynthetic pathway for N-linked glycoproteins
and GPI-anchored proteins.
1. Roos, J., Sternglanz, R., and
Lennarz, W. J. (1994) Proc. Natl. Acad. Sci., U. S. A., 91, 1485-1489
P-6
NEW MATERIALS AND TECHNIQUES FOR GLYCOMEDICINE
Toshiyuki Inazu
The Noguchi Institute, Tokyo, Japan
Glycoconjugates
play an important role in biological processes, such as cell recognition,
cell adhesion, immunogenic recognition, and so on. We have reported
the chemo-enzymatic synthesis of glycopeptide having natural oligosaccharide.1)
We describe herein the new materials and techniques for the preparation
of glycomedicine.
First,
we found Fmoc-Asn(Sugar)-OH reacted to the amino group in some drug using
PyBOP reagent in NMP. Fmoc-Asn(Sugar)-OH was prepared from H-Asn(Sugar)-OH
from ovalbumin or egg yolk in high yield.2) For instance we designed
and synthesized the cyclodextrins (CDs) having natural oligosaccharides
as targeting drug delivery system carrier. This oligosaccharide-CD
conjugate was obtained by the reaction of 6-mono-amino-b-CD and Fmoc-Asn(Sugar)-OH.
High-mannose-type oligosaccharide-branched CD were adopted for dual recognition
both for oligosaccharide-recognition with immobilized concanavalin A and
for inclusion association with immobilized cholic acid as a model drug
using an optical biosensor apparatus based on surface plasmon resonance.3)
Next, we
also designed and synthesized GlcNAc-OCH2COOH as a new chemo-enzymatic
protein modifier. We tried the modification reaction of bovine insulin
using this modifier, and obtained the modified insulin having _GlcNAc sugaring
tag_ at the Lys side chain. After enzymatic transglycosylation reaction
using endo-b-N-acetylglucosaminidase from Mucor hiemalis ( endo-M ),1)
we obtained the _neo-insulin_ having natural oligosaccharide.
1. Mizuno M., Haneda K., Iguchi R.,
Muramoto I., Kawakami T., Aimoto S., Yamamoto K., and Inazu T. (1999) J.
Am. Chem. Soc., 121, 284-290.
2. Inazu T., Mizuno M., Yamazaki
T., and Haneda K. _Peptide Science 1998_ Ed by Kondo M. (1999) Protein
Research Foundation, Osaka, pp. 153-156.
3. Matsuda K., Inazu T., Haneda
K., Mizuno M., Yamanoi T., Hattori K., Yamamoto K., and Kumagai H. (1997)
Bioorg. Med. Chem. Lett., 7, 2353-2356.
P-7
CHANGES IN GLYCOSYLATION AND ACTIVATION OF VITRONECTIN, AN
EXTRACELLULAR MATRIX GLYCOPROTEIN, DURING LIVER REGENERATION. -
EFFECT OF GLYCOSYLATION ON MULTIMERIZATION OF VITRONECTIN ANALYZED
BY ANALYTICAL ULTRACENTRIFUGATION -
Kimie Asanuma1, Fumio Arisaka2 and
Haruko Ogawa1
1Graduate School of Humanities and
Sciences, Ochanomizu University, Tokyo, and 2Graduate School of Bioscience
and Biotechnology, Tokyo Institute of Technology, Tokyo, Japan
Vitronectin (VN), a multifunctional
glycoprotein present in the extracellular matrix and plasma , plays a role
in cell adhesion, cellular motility and matrix remodeling. VN binds to
various matrix ligands, such as integrins, collagens, type 1 plasminogen
activator inhibitor and urokinase receptor, through its conformational
transition the native inactive form to an active form. Some of these binding
activities are affected by changes in glycosylation of VN1). For
example, the collagen-binding activity is enhanced by decreased glycosylation
of VN during liver regeneration after a partial hepatectomy2).
In the present study, effect of
glycosylation on multimerization of VN was analyzed by analytical untracentrifugation
to elucidate the modulation mechanism of the biological activity by glycans.
Sedimentation velocity analysis at pH 4.5, an optimum pH for collagen binding,
indicated that multimerization of VN was more remarkable than that at pH
7.5. Neuraminidase-treated VN formed a multimer larger than
that of VN incubated without the enzyme. N-glycanase treatment gradually
increased the size of the VN multimer. The stability of VN multimer
against the denaturing agent was measured by sedimentation equilibrium
analyses at various concentrations of guanidine-HCl. The neuraminidase-treated
or N-glycanase-treated VN remained in a multimer form at a guanidine-HCl
concentration in which control VN dissociated into monomers, enhancing
thus the multimer stability of deglycosylated VNs. These results
suggest that deglycosylated VN increased the size and stability of the
multimer to eventually enhance the collagen binding activity by multivalent
effect.
1) Yoneda et al., (1998) Biochemistry
37, 6351-6360.
2) Uchibori-Iwaki, H., Yoneda,
A., et al., (2000) Glycobiology 10, in press
P-8
BIOLOGICAL SIGNIFICANCE OF GLYCOSYLATION CHANGE IN VITRONECTIN DURING
LIVER REGENERATION AND INJURY
Risa Suzuki1, Sadako Yamada2, Haruhi
Uchibori-Iwaki1, Mayumi Tanabe1, Sachie Oda-Tamai3, Shigemi Kato3, Nobu
Akamatsu3, Atsuko Yoneda1, 4 and Haruko Ogawa1
1Graduate School of Humanities and
Sciences, Ochanomizu University, Tokyo, 2Faculty of Medicine, Tottori University,
3St. Marianna University Medical School, Kawasaki, and 4National Institute
of Bioscience and Human-Technology, Tsukuba, Japan
Vitronectins (VN) are multifunctional
adhesive glycoproteins that originate mainly in hepatocytes and circulates
in the blood stream. VN are also present in the extracellular matrix
of most tissues and play a central role in matrix remodeling through the
interactions with various matrix ligands, such as integrins, collagens,
type 1 plasminogen activator inhibitor and urokinase receptor. The collagen
binding activity is affected by the presence or absence of N-glycan covalently
linked to VN1）. However, in chronic liver diseases, the collagen-binding
VN in plasma has been reported to increase and correlate with certain fibrous
markers2). To elucidate the glycan modulation, changes in VN during liver
regeneration after partial hepatectomy, during hepatic injury, or in a
diseased state were studied.
Plasma concentrations of VN declined
in rats during liver regeneration 24 h after partial hepatectomy, while
carbohydrate concentrations of VN decreased to 2/3 that of sham-operated
rats. Carbohydrate composition and lectin reactivity indicated that the
N-glycan structures and sialylation significantly changed without affecting
the peptide portion after partial hepatectomy. VN from partially hepatectomized
rats were found to exhibit markedly enhanced binding to type I collagen.
The enzymatic deglycosylation of VN demonstrated that collagen binding
increased by 1.2 times after deN-glycosylation of VN, while it increased
by more than 2.9 times after desialylation3）. The plasma levels of VN
significantly declined in rats with CCl4-induced injury and in humans with
chronic liver diseases such as hepatitis, cirrhosis and hepatocellular
carcinoma with cirrhosis. Carbohydrate analyses of VN from rats with CCl4-induced
injury and humans with liver cirrhosis revealed that fucosylation and branching
of N-linked oligosaccharides were considerably promoted. These findings
suggest that the collagen binding activity of VN is modulated by the alteration
of peptide glycosylation during liver regeneration after partial hepatectomy
and during the pathological processes, which may contribute to the tissue
remodeling processes.
1. Yoneda et al., (1998) Biochemistry
37, 6351-6360.
2. Yamada, S., Kobayashi,
J., et al., (1996) Clin. Chim. Acta 252, 95-103.
3. Uchibori-Iwaki, H., Yoneda,
A., et al., (2000) Glycobiology 10, in press.
P-9
DETECTION OF TISSUE SPECIFIC SUGAR CHAINS BY 2-DIMENSIONAL HPLC SUGAR
MAPPING OF PYRIDYLAMINATED SUGAR CHAINS
Shin-ichi Nakakita1, Kazuhiro Ikenaka2,
and Sumihiro Hase1
1Department of Chemistry, Graduate
School of Science, Osaka University, Osaka, and 2National Institute for
Physiological Sciences, Okazaki National Research Institutes, Aichi, Japan
We have developed a
fluorescence labeling method of sugar chains and the method was coupled
with 2-dimensional HPLC sugar mapping1). The method provide an excellent
resolution of sugar chains, which enables the identification of a large
numbers of sugar chains. A part of a tissue was freeze-dried without
any pretreatment, and the sample was treated with anhydrous hydrazine (at
100C for 10 h). The sugar chains liberated were N-acetylated and
the products were fluorescence labeled (pyridyl-amination). The excess
reagents were removed by gel filtration. The PA-sugar chains were
first separated by size-fractionation HPLC using PA-isomaltooligo-saccharides
as an elution scale. The fractions thus obtained were further separated
by reversed-phase HPLC (2-dimensional HPLC sugar mapping). A freeze-dried
sample of 2 mg was enough to make a 2-dimensional sugar map. Using the
map, more than 200 sugar chains could be separated. Using this principle,
we have detected brain-type sugar chains, when the differential display
of sugar chains were done by comparing the 2-dimensional sugar maps obtained
from several mouse tissues. Two brain-specific sugar chains (BA-1
and BA-2) were detected in mouse neural tissues and their structures were
determined to have the following chemical structures: BA-1, GlcNAcb1-2Mana1-6(GlcNAcb1-4)(Mana1-3)Manb1-4GlcNAcb1-4(Fuca1-6)GlcNAc;
BA-2, GlcNAcb1-2Mana1-6(GlcNAcb1-4)(GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1-4(Fuca1-6)GlcNAc2).
The two brain-specific sugar chains were found to be developmentally regulated,
and glycoproteins with the BA-1 and BA-2 structures were enriched in the
membrane fraction. The period of rapid BA-2 up-regulation in the
cerebrum coincided with the stage when neurons formed synapses. Judging
from these results, it is possible that sugar chains are isolated from
a small part of a tissue and analyzed by 2D-HPLC mapping of PA-sugar chains.
1. Hase, S. (1994) Methods
in Enzymology, 230, 225-237
2. Shimizu, H., Ochiai, K.,
Ikenaka, K., Mikoshiba, K., and Hase. S. (1993) J. Biochem., 114, 334-338
P-10
THE ROLE OF PROTEIN KINASES IN GLYCOPROTEIN GMP-140 EXPRESSION ON
ACTIVATED HUMAN PLATELETS
Masaru Shoji1, Ryoko Kudo2, Moe Wakui1,
Mari Nakano2, Shoji Tsutaya2,
Junko Saito2, Hideetsu Takamatu2,
Minoru Yasujima1
1Department of Laboratory Medicine,
Hirosaki University School of Medicine, Hirosaki, and 2Department of Clinical
Laboratory, Hirosaki University Hospital, Hirosaki, Japan
Glycoprotein GMP-140 (CD62P) is one
of surface markers expressed on platelet membrane when activated.
CD62P plays an important role in the platelet aggregation. Arginine
vasopressin (AVP) is known to activate platelets via AVP V1 receptors.
However, downstream event of the stimulation needs further clarification.
Therefore, we explored intracellular signaling pathway responsible
for membrane expression of CD62P after AVP stimulation.
[Methods]
Platelet rich plasma and whole peripheral
blood from normal subjects were used in the present study. Selection
of activated platelet by expression of CD62P was made according to the
simultaneity of the stainings with CD42b antibody as well as by the use
of a precise gating on the flow cytometer. These expressions of the
surface markers were compared with AVP induced platelet aggregation measured
by conventional photometric method. Effects of inbitors and
stimulators for protein kinase A (PKA) and protein kinase C (PKC) were
determined.
[Results]
Serial doses of AVP (10, 100, 1000,
and 10000 nM) provoked platelet aggregation and CD62P expression in a dose
dependent manner. Staurosporine, a PKC inhibitor, inhibited platelet
aggregation and CD62P expression. Whereas phorbol ester (PMA), a
PKC stimulator, itself provoked platelet aggregation and CD62P expression.
It also strengthened the AVP induced platelet activation. On the
other hand, 8-Br-cAMP, a membrane permeant cAMP analogue, was without effects
on platelet activations. PMI, a PKA inhibitor, had a little effect
on them.
[Conclusion]
From these results, it is
suggested that PKC plays a dominant role in the AVP actions on platelet
aggregation and CD62P expression.
P-11
ANALYSIS OF p-NITROPHENYL N-ACETYL-α-D-GALACTOSAMINIDE-INDUCED
OLIGOSACCHARIDES PRODUCED BY THE CULTURED HUMAN COLON
ADENOCARCINOMA CELL, DLD-1
Akihiko Matsuki, Junko Kukidome,
and Akihiro Munakata
First Department of Internal Medicine,
Hirosaki University School of Medicine, Hirosaki, Japan
Mucus glycoproteins
(mucins) are high molecular weight
O-linked glycoproteins and they
have important biological functions. In order to clarify the mechanism
of elongation of O-glycan chains, p-nitrophenyl
N-acetyl-α-D-galactosaminide
(GalNAc-PNP) was added to the medium of a cultured human colon adenocarcinoma
cell line, DLD-1.
After incubation for 7 days
GalNAc-PNP-induced oligo- saccharides were derived from the medium.
These were concentrated, purified and separated by gel-filtration, ion-exchange
high performance liquid chromatography (HPLC) and reverse-phase HPLC. Following
separation, the GalNAc-PNP-induced oligosaccharides were subjected to carbohydrate
composition analysis and enzymic digestion analysis. Some kinds of
GalNAc-PNP-induced oligosaccharides were identified. Because DLD-1
produced galactosyl (Gal)-GalNAc-PNP but not N-acetylglucosaminyl
(GlcNAc)-GalNAc- PNP, and produced considerably more branched Gal-(GlcNAc-)
GalNAc-PNP than unbranched GlcNAc-Gal-GalNAc-PNP, it was considered that
N-acetylglucosamine binds to N-acetylgalactosamine after initial
binding of galactose to N-acetylgalactosamine. It is also
likely that further elongation of O-glycan chains is inhibited by
the binding of sialic acid or sulfate to N-acetylgalactosamine.
In summary, sugar chain
elongation analysis of GalNAc-PNP in cultured DLD-1 cells is a potentially
useful method for elucidating the biosynthetic mechanisms underlying the
production of O-linked glycoproteins.
1) Hirosaki Medical Journal, in press
P-12
CONVENIENT METHOD FOR STRUCTURAL DETERMINATION OF CELL SURFACE
GLYCANS RELATED TO CANCER AND OTHER DISEASES
Ikuko Ishii-Karakasa
Department of Biochemistry, School
of Medicine, Kitasato University, Kanagawa, Japan
Leukosialin, glycophorin A, submaxillary
mucin, intestinal mucin, epitectin, fetuin etc. are glycoproteins, which
have some sialyl oligosaccharides. The structure of the oligosaccharide
moiety in these glycoproteins has been partially clarified, and changes
in the sialyl oligosaccharide with differentiation, malignant transformation
and immunodeficiency have been extensively studied.
Because there is an only small quantity
of cell surface glycans related to these diseases, it is very important
to be able to determine the complete structures of oligosaccharides with
a small quantity on glycoconjugates. Various methods have recently been
developed for the analysis of oligosaccharides in glycoproteins, and structural
analysis with high sensitivity can be performed today within a very short
period of time. As one of such methods, monosaccharide and/ or oligosaccharide
is analyzed with high performance liquid chromatography (HPLC), and the
need for a method of structural determination of oligosaccharides by 1H-NMR
spectroscopy became evident during the course of the investigation of glycoprotein
in biological systems. However, the 1H-NMR spectra of oligosaccharides
are complicated, because most signals of the component sugars of the oligosaccharide
appear in the region of 3.4 - 4.6 ppm.
In the present study, convenient
method for the analysis of oligosaccharides related to diseases using HPLC
and 1H-NMR spectroscopy was developed. Regarding the 1H-NMR spectroscopy,
development of the new structural analysis to assign all the chemical shifts
and the coupling constants in the oligosaccharide with a small quantity
was performed. Since 1H-NMR spectra of oligosaccharides are assumed to
observe as a superposition of the spectra of each component sugar, it has
led to the suggestion that the coupling constants of each component sugar
of the oligosaccharide are temporarily substituted for that of the corresponding
monosaccharides. Therefore, using fetuin as model compound and other oligosaccharides
related to diseases, it has been found that the best chemical shifts and
coupling constants are determined by repetition of spectral simulation
using the coupling constants of the corresponding monosaccharides. These
data can consequently be utilized for the analysis of a spectrum surveyed
by different spectrometer frequencies.
P-13
STRUCTURAL ANALYSIS OF THE OLIGOSACCHARIDE UNITS OF XYLOGLUCAN AND
THEIR EFFECTS ON GROWTH OF COLO 201 HUMAN TUMOR CELLS
Yoji Kato1, Junko Uchida1, Seiko
Ito1, and Yasushi Mitsuishi2
1Laboratory of Food Science, Faculty
of Education, Hirosaki University, Hirosaki, 2National Institute of Bioscience
and Human Technology, AIST, Tsukuba, Japan
Xyloglucans are major
structural polysaccharides of the primary cell walls of all higher plants.
A marked characteristic of the xyloglucans is that the polysaccharides
consist of a linear backbone chain of 1,4-linked β-D-glucopyranosyl
residues and side chains of single α-D-xylopyranosyl
residues, β-D-galactopyranosyl-(1→2)-α-D-xylopyranosyl
residues, α-L-fucopyranosyl-(1→2)-β-D-galactopyranosyl-(1→2)-α-D-xylopyranosyl
residues, etc, that are attached to the C-6-positions of the bakbone chain.
Treatment of xyloglucan with endo-β-1,4-glucanase
results in the cleavage of unbranched 4-linked glucopyranosyl residues
in the xyloglucan backbone, generating oligosaccharide subunits of the
xyloglucan polymer. Xyloglucan polysaccharides and some oligosaccharides
play a role of the regulation of plant growth. Furthermore, xyloglucan
oligo- and polysaccharides were demonstrated to have some physiological
effects on animals. From this point of view compositional analysis of
oligosaccharide units in xyloglucan polymer is very important.
The oligosaccharide
units of xyloglucans isolated from the cell walls of commercially available
vegetables1)
and fruits were comparatively analyzed by enzymatic digestion followed
by anion-exchange chromatography with pulsed amperometric detection2).
The results indicated a high degree of regularity in the arrangement of
unsubstituted glucopyranosyl residues along the β-glucan
backbone in angiosperm xyloglucans, and no regularity in that in Gramineae
xyloglucan.
In addtion, different
type of xyloglucan oligo- and polysaccharides were tested for effects on
the growth of COLO 201 human tumor cells. The cell growth was reduced by
fucose and/or galactose-containing oligo- and polysaccharides. This suggests
that the growth inhibition depends on the structure of side chains of xyloglucans.
1. Konishi, T., Mitsuishi ,Y., and
Kato ,Y. (1998) J. Appl. Glycosci., 45, 401-405
2. Konishi, T., Mitsuishi ,Y., and
Kato ,Y. (1998) Biosci. Biotechnol. Biochem., 62, 2421-2424
P-14
FUNCTIONAL ANALYSIS OF CELLULOSE-SYNTHASE-LIKE GENES IN ARABIDOPSIS
THALINA
Teruko Konishi, Fukumi Sakai, and
Takahisa Hayashi
Wood Research Institute, Kyoto University,
Gokasho, Uji, Kyoto, Japan
A putative gene for celluloseﾐsynthase-like
4-b-glucosyltransferase
was isolated from arabidopsis, cotton and rice as a homologous gene of
acetobacter BcsA and arabidopsis RSW1. Large numbers of genens are being
discovered on a daily basis for a variety of organisms including Arabidopsis
thaliana. Cellulose-synthase-like genes have been found more than 40 genes
in arabidopsis. We believe that some of these genes encode other related
glycosyltransferases, rather than that all the genes are required for tissue-specific
expression. In order to define and identify the activity of the genes products,
we have developed a heterogeneous expression system in Pichia pastoris
and COS cells. This system is useful to determine directly the activity
of callose, xyloglucan, xylan or cellulose synthase from the recombinant
proteins, and also to examine the interaction between sucrose synthase
and glucosyltransferase during glycan synthesis. The expression vectors
constructed contains a complementary T base for TA cloning at the cloning
site and signals for secretion and antibody recognition attached to the
structural gene inserted. In an attempted expression study for sucrose
glucosyltransferases cDNA, the gene product was detedted by Western blot
analysis and showed sucrose synthase activity. This technology provides
new functional information on cellulose-synthase-like genes.
P-15
PROTEOGLYCANS IN THE NUCLEUS PULPOSUS OF CANINE INTERVERTEBRAL DISCS
AFTER CHONDROITINASE ABC TREATMENT
Atsushi Ono, Taisuke Nitobe and Seiko
Harata
Departments of Orthopedics, Hirosaki
University School of Medicine, Hirosaki, Japan
Recently, chondroitinase ABC
(CABC) has attracted attention as a possible replacement enzyme for chymopapain
(CP)1). In this study, the proteoglycans (PGs) changes of the nucleus pulposus
after degradation of glycosaminoglycan (GAGs) by CABC or core protein by
CP, were examined using X-ray, MRI, histological and biochemical techniques.
Lumbar intervertebral discs
of 35 beagles were injected with either CABC or CP. A comparative study
was made in regards to the following: X-ray and MRI were taken in order
to examine changes of disc spaces and the disc water content. Sagittal
serial sections at the disc level were stained in H-E for light microscopic
examination. Biochemical analysis using high-performance liquid chromatography
was carried out in order to examine quantity, molecular weight, acidity
of PGs and chain length of GAGs.
The disc space narrowing, the decrease
of disc water content and the degeneration of chondrocyte and matrix in
nucleus pulposus of CABC were milder than that of CP. The quantity of PGs
after degradation by CABC was the same as that of CP. However, the molecular
weight of PGs after degradation by CABC was bigger than that of CP. The
chain length of GAGs after degradation by CABC was longer than that of
CP. The acidity of PGs after degradation by CABC was lower than that of
CP.
Similarly to CP, CABC degrades
PGs in the nucleus pulposus and decreased PGs quantity 2). However, the
quantity, molecular weight and acidity of re-synthesized PGs, and chain
length of re-synthesized GAGs after degradation were very different between
the two enzymes. The difference between the two enzymes in regard to disc
space narrowing and changes of the disc water content over time might
result from the differences of characters of re-synthesized PGs.
1. Ono, A., Harata, S., Takagaki,
K., and Endo, M. (1998) J. Spinal Disorder, 11, 253-260
2. Nitobe, T., Harata, S., Okamoto,
Y., Nakamura, T., and Endo, M. (1988) Spine, 13, 1332-1339
P-16
THE ROLE OF PROTEOGLYCANS IN OSSIFICATION OF SPINAL LIGAMENT
Masahiro Yukawa, Taito Itabashi,
Kazunari Takeuchi, Hozumi Narita, Yusuke Takeda, Akihiro Okada, Kazumasa
Ueyama, and Seiko Harata
Department of Orthopedics，Hirosaki
University School of Medicine, Hirosaki, Japan
Proteoglycan is present in the extracellular
matrix of various connective tissues. It is considered to have important
functions that influence the properties of extracellular matrix.
In this study, proteoglycan was
purified from human yellow ligaments by ion-exchange and gel-filtration
chromatography. Age-related changes in proteoglycan and glycosaminoglycan
chains were studied using specimens obtained from patients divided into
four age groups. The structural differences between proteoglycan from normal
and ossified ligaments were analyzed. Small proteoglycan was separated
into three types by hydrophobic chromatography.
Small proteoglycan was present in
similar amounts in all groups. Conversely, large proteoglycan increased
with aging. Glycosaminoglycan in large proteoglycan consisted mainly of
chondroitin 6-sulfate, whereas in small proteoglycan it consisted mainly
of dermatan sulfate, although the ratio of chondroitin 6-sulfate increased
with aging. In the chains composed mainly of dermatan sulfate, glucuronic
acid replaced L-iduronic acid near the carbohydrate-protein linkage region,
and the proportion of 6-O-sulfated N-acetylgalactosamine increased. The
major components of glycosaminoglycan chains of normal and ossified yellow
ligaments were dermatan sulfate and chondroitin sulfate, respectively.
Also, affinity HPLC on hydroxyapatite columns showed that dermatan sulfate
chains bound to the hydroxyapatite more strongly than did chondroitin sulfate
chains.
Small proteoglycans separated by
hydrophobic chromatography were identified as decorin, decorin-subtype,
and biglycan by analyses with immunoassay, amino acid sequencing, and HPLC
etc. The molecular size of both decorin and decorin-subtype was 80 kDa,
and that of biglycan was 200 kDa. The N-terminus amino acid sequence of
decorin-subtype corresponded with that of decorin, but it was different
from decorin in the following: 1) difference of behavior in hydrophobic
chromatography; 2) no response in Western blotting immunoassay using anti-human
decorin antibody; and 3) difference of glycosaminoglycan in compositions.
The glycosaminoglycan chains bound to core protein of the decorin-subtype
were the same size as those of decorin, but were mainly dermatan sulfate,
whereas those of decorin were mainly chondroitin/dermatan sulfate. Interaction
between small proteoglycans and elastin was investigated with a surface
plasmon resonance biosensor. Decorin-subtype had affinity with elastin,
whereas decorin and biglycan did not.
These results indicate that the
change from dermatan sulfate to chondroitin sulfate is important in age-related
change and ossification of ligaments, because chondroitin sulfate chains,
which have lower affinity for hydroxyapatite, might contribute to the facilitation
of the crystallization of hydroxyapatite. Likewise, decorin-subtype, which
has mainly dermatan sulfate chains, is considered to be important for the
maintenance of a normal extracellular matrix in human yellow ligament.
1. Okada, A., Harata, S., Takeda,
Y., Nakamura, T., Takagaki, K. and Endo, M. (1993) Spine 18, 2261-2266
2. Takeda, Y., Harata, S., Takagaki,
K., Narita, H. & Endo, M. (1992) Connect. Tissue 24, 109-114
P-17
A NOVEL 4-METHYLUMBELLIFERYL-b-D-XYLOSIDE
DERIVATIVE,
SULFATE-O-3-XYLOSYLb1-(4-METHYLUMBELLIFERONE),
ISOLATED FROM
CULTURE MEDIUM OF HUMAN SKIN FIBROBLASTS, AND ITS ROLE IN
METHYLUMBELLIFERONE-INITIATED GLYCOSAMINOGLYCAN BIOSYNTHESIS
Toshiyuki Tazawa, Hideki Matsuya,
Daisuke Kudo, and Mutsuo Sasaki
Second Department of Surgery , Hirosaki
University School of Medicine, Hirosaki, Japan
Although the mechanisms
involved in glycosaminoglycan (GAG) biosynthesis are not yet fully understood,
it has been demonstrated that GAG biosynthesis is initiated by the transfer
of a xylose residue from UDP-Xyl to a serine residue of the core protein,
followed by stepwise addition of individual monosaccharides from UDP-sugars
by a series of glycosyltransferase reactions. It has been reported that
addition of a ｧ-xyloside, such as p-nitrophenyl-ｧ-D-xyloside, 4-methylumbelliferyl-ｧ-D-xyloside
(Xyl-MU) to cell culture medium induces elongation of GAG chains, which
is initiated by the ｧ-xyloside acting as a primer.
In the present study,
human skin fibroblasts were cultured in the presence of Xyl-MU and Xyl-MU
induced oligosaccharides (Gal-Gal-Xyl-MU, Gal-Xyl-MU, Sia-Gal-Xyl-MU, GlcA-Xyl-MU,
sulfate-GlcA-Xyl-MU and Xyl-Xyl-MU) were synthesized. A novel Xyl-MU derivative
was obtained, in addition to the previously reported Xyl-MU derivatives.
This Xyl-MU derivative was subjected to carbohydrate composition analysis,
enzyme digestion, Smith degradation and ion-spray mass spectrometric analysis
and the results indicated that it was sulfate-0-3-Xyl-MU. This Xyl-MU derivative
was also synthesized when Xyl-MU was incubated with [35S]PAPS. But when
Gal-Xyl-MU and Gal-Gal-Xyl-MU were incubated with [35S]PAPS, incorporation
of [35S]sulfate from [35S]PAPS into either galactose or xylose was not
observed. When Xyl-MU or sulfate-Xyl-MU was incubated with UDP-[3H]Gal,
incorporation of [3H]Gal into Xyl-MU was observed, but incorporation into
sulfate-Xyl-MU was not.
These results indicate
that chain elongation from Xyl-MU is inhibited by sulfation of Xyl-MU,
and that Xyl-MU sulfation is involved in the control of Xyl-MU-initiated
glycosaminoglycan biosynthesis.
1. Tazawa, T., Takagaki, K., Matsuya,
H., Nakamura, T., Sasaki, M., and Endo, M. (1998) Glycobiology., 8, 879-884
2. Takagaki, K., Nakamura, T., Kon,
A., Tamura, S., and Endo, M. (1991) J. Biochem., 109, 514-519
P-18
A BINDING BETWEEN CALCIUM AND CHONDROITIN SULFATE CHAINS OF SALMON
NASAL CARTILAGE GLYCOSAMINOGLYCAN
Hidemitsu Uchisawa1, Bun-ichi Okuzaki2,
Junji Ichita1, Hajime Matsue1
1Division of Biotechnology, Aomori
Adnanced Industrial Technology Center, Aomori,
and 2Yamaishi Co. Ltd., Aomori,
Japan
Glycosaminoglycan, chondroitin
sulfate(ChS), was isolated from the nasal cartilage of salmon, Oncorhynchus
keta. The ChS were obtained by the method of Actinase E digestion, DEAE
Sephadex A-50 ion-exchange chromatography and Sephacryl S-300 gel filtration.
They gave a single band on the electrophoresis using cellulose acetate
membrane and contained equimolar ratios of GlcA and GalNAc. After chondroitinase-ABC
degradation of the chondroitin sulfate, the products were analyzed by a
weak anion-exchange resin on a high-performance liquid chromatography system
to give a following results, DDi-0S, DDi-4S, DDi-6S = 8 : 31 : 61.
On the other hand,
it was known that chondroitin sulfates distributed in all connective tissues
like cartilage of invertebrates and vertebrates, and varied in its quantity
and quality with maturation of living. It has been noted that the character
of calcium binding was important one as the biological function of chondroitin
sulfates. In 1968, Woodward et al.1) reported that bone calcification was
happened by the release of calcium bound to proteoglycan in cartilage.
In this work, we examined that which functional groups of carboxyl and
sulfate of chondroitin sulfates bind to calcium by analyses using 1H-NMR
and FT-IR.
As the results, we
found that anomeric protons at GlcA and GalNAc of chondroitin sulfate were
shifted to higher field and more clearly splitted at 1H-NMR spectra depend
on concentration of calcium ion bound, but not in the case of sodium ion.
And we speculated that chondroitin sulfates bound selectively to calcium
ion in the presence of both calcium and sodium ions. Furthermore, these
speculations were confirmed by photometric quantitative analysis of calcium
ion using Arsenazo-III.
1. C. Woodward et al. (1968) Proc.
Nat. Acad. Sci., 60, 515
P-19
A SIMPLE METHOD FOR EXTRACTION OF PROTEOGLYCAN AND ITS MEDICAL
APPLICATION
Mitsuo Majima1, Keiichi Takagaki2,
Shin-ichiro Sudo2, Syuichi Yoshihara3, Yoshiaki Kudo1, and Shohei Yamagishi1
1Kakuhiro Co. Ltd., 2Department of
Biochemistry, and 3Second Department of Surgery, Hirosaki University School
of Medicine, Hirosaki, Japan
Proteoglycans, which occur in most
mammalian tissues, are widely present on cell surfaces and in the extracellular
matrix. They are thought to have a range of physiological functions including
water holding capacity. Therefore, we performed a fundamental study with
the aim of developing a simple method for extraction of proteoglycan and
applying then to medical uses.
Nasal cartilage obtained
from salmon was extracted for 48 h at 4�C with 10 vol. of 4% acetic acid.
From the extract, the nasal-cartilage proteoglycan of salmon was purified
by ethanol precipitation and dialysis using membrane of molecular weight
cut off value of one million. By this method, 240 mg of proteoglycan
was recovered from 50 g of the nasal cartilage. The proteoglycan
had a molecular size of 344 kD, and contained 7% protein and a very large
number of chondroitin sulfate.
Moreover, the proteoglycan
was administered orally to 11-week-old male mice with experimental enterocolitis
(dextran sulfate sodium salt induced). Then, the time course of survival
rate, body weight and pathologic findings were observed and measured.
In the administration group, the survival rate increased significantly
and no decrease of body weight was observed in comparison with that in
the control group. Consequently, the onset of dextran sulfate sodium
salt induced enterocolitis was inhibited by administration of the nasal-cartilage
proteoglycan from salmon, which will be useful clinically as a new drug
for inflammatory bowel disease.
P-20
PHOTOCROSSLINKABLE CHITOSAN: AN EFFECTIVE ADHESIVE WITH SURGICAL
APPLICATION
Masayuki Ishihara1, Katsuaki Ono2,
Yoshio Saito3, Hirofumi Yura3, Hidemi Hattori1, Takemi Matsui1, and Akira
Kurita1
1Division of Biomedical Engineering,
Research
Institute, National Defense Medical
College, Tokorozawa, 2Department
of Surgery II, National Defense Medical College,
Tokorozawa, and 3NeTech Inc., Japan
Chitin is a linear homopolymer
of 1,4b-linked
N-acetyl-D-glucosamine, and chitosan is partially
N-deacetylated chitin. Chitosan already has been proposed as a biomedical
material. It has been served to accelerate wound healing and has
a hemostatic potential. In addition, chitosan is biodegradable
and nontoxic. Chitosan also is known for its immunologic activity,
that is, its macrophage activation, its cytokine production, and its inhibition
of infection.
We designed a new photocrosslinkable
chitosan molecule (Az-CH-LA) that contains both lactose moieties and photoreactive
azide groups and may be used as a biological adhesive for medical purpose.
Introduction of the lactose moieties resulted in a much more water-soluble
chitosan at neutral pH. Application of ultraviolet light (UV) irradiation
of Az-CH-LA produced an insoluble hydrogel like a soft rubber within 60
s.
In the present work,
the sealing ability of the chitosan gel has been evaluated as a bursting
pressure, using removed aorta, trachea and lung of farm pigs. The
sealing ability of the chitosan gel has been found similar or even stronger
than that of fibrin glue which is widely used in clinics. In in vivo
experiment using rabbits, all rabbits whose cartid artery (n=8) or lung
(n=8) have been punctured with needle and then sealed with the chitosan
gel stayed alive during the one month period without bleeding or air leakage
from the punctured site. Histological examinations have demonstrated
that a fraction of the chitosan gel 30 days after applying has been phagocytosed
by moacrophages, partially degraded and inducing the formation of fibrous
tissue around the hydrogel. These results suggest that the photocrosslinkable
chitosan developed here has the potential of serving as a new tissue adhesive
in surgical uses.
P-21
EFFECTS OF HYALURONAN ON MATRIX METALLOPRO- TEINASE ACTIVITIES IN
HUMAN SKIN FIBROBLASTS
Toshiya Nakamura1, 3, Asami Sakamoto2,
and Takashi Ishikawa2, 3
1Department of Nursing, 2Department
of Medical Technology, Hirosaki University School of Allied Medical Sciences,
and 3Department of Medical Technology, Hirosaki University School of Health
Sciences, Hirosaki, Japan
It has been reported
that hyaluronan plays an important role in embryonal development, inflammation,
wound healing, angiogenesis and tumorigenesis. Although activation
or inhibition of matrix metalloproteinases (MMPs) has also been observed
in such conditions, the relation between hyaluronan and MMP activities
are not fully investigated. Therefore, effects of hyaluronan
on the activity of MMPs were examined in human skin fibroblasts.
Quiescent subconfluent
cells were cultured for 2 days in the presence of hyaluronan with various
sizes (Mr=1200 k, 800 k, 300 k, 100 k and 40 k). The conditioned
media were collected and treated with Streptomyces hyaluronidase.
The fractions precipitated with 80% saturation of ammonium sulfate were
collected from the media, and used as enzyme preparation. The activities
of MMPs were examined by gelatin- or casein zymography under denaturing
but nonreducing conditions.
Gelatin zymography
showed an increase of MMP-2 (gelatinase A) activity especially by higher-molecular
weight hyaluronan preparation (1200 k, 800 k and 300 k).
MMP activity appeared as 47 kDa band was also increased in the same way.
On the contrary, casein zymography showed a decrease of MMP-3 (stromelysin-1)
activity in the presence of hyaluronan, regardless of its chain sizes.
These results suggest
that hyaluronan have some effects on the activity of MMPs and may be linked
to the proteolytic tissue remodeling in various physiological and pathological
conditions.
P-22
HYALURONAN KNOCKDOWN EXTRACELLULAR MATRIX OF CULTURED HUMAN SKIN
FIBROBLAST BY USING OF 4-METHYLUMBELLIFERONE
Yasufumi Endo1, Masaru Funahashi2,
Ikuko Kakizaki2, Keiichi Takagaki2 Masahiko Endo2, Gen Takahashi3, and
Masaru Yokoyama1
1Department of Pediatrics, 2Department
of Biochemistry, and 3Department of Anatomy, Hirosaki University School
of Medicine, Hirosaki, Japan
Hyaluronic acid (HA)
is an ubiquitous glycosaminoglycan consisting of glucuronic acid and N-acetylglucosamine,
and is found in almost all of the extracellular matrix (ECM). It participates
in interaction with numerous matrix molecules. Its function is now known
not only structural function such as supporting the cells and providing
tissue stability, but also physiological function such as cell attachment
and adhesion.
Nakamura et al.1) reported
that 4-methylumbellyferone (MU) inhibits HA synthesis in cultured human
skin fibroblasts (HSF). HA deficient ECM can be made by using of culture
medium which MU is added. It is useful for studying of the function of
HA such as interaction of other ECM molecules.
HSF were cultured to
confluency. MU was added to the culture medium at various concentration,
and cells were incubated for 3 days. Then its medium and the cell layer
was collected. Type I collagen, fibronectin and CD44 were mesured with
enzyme immunoassay. Proteoglycan was measured as [35S]-sulfate uptake.
Electromicroscopy of ECM was also performed.
HA concentration of
each medium and cell layer was mesured. Contents of HA on medium and cell
layer was decreased in the present of MU. Compared with control, the concentration
of type I collagen and proteoglycan were increased in the medium in the
present of MU. The concentration of proteoglycan, fibronectin and CD44
were decreased in the cell layer in the present of MU. Electron microscopy
of HSF cultured in the presence of MU was performed. Cell layer which incubated
with medium containing 0.5 mM MU had poor ECM structure, and had very narrow
intra cellular space.
This study showed that
cultivation of HSF in the presence of MU led to the formation of HA deficient
ECM. No such model has yet been found for HA. This model must be useful
for functional studies of HA in ECM. It would be distinced that the role
of HA at ECM for transplantation.
1. Nakamura, T., Takagaki, K., Shibata,
S., Tanaka, K., Higuchi, T., and Endo, M. (1995) Biochem. Biophys.
Res. Commun., 208, 470-475
2. Endo, Y., Takagaki, K., Takahashi,
G., Kakizaki, I., Funahashi, M., and Yokoyama, M. in Progress in Transplantation,
edited by Munakata, A., and Yokoyama, M., Elsevier Science B. V., Amsterdam,
the Netherlands, in press
P-23
ECHANISM OF THE INHIBITION OF HYALURONAN SYNTHESIS BY
4-METHYLUMBELLIFERONE
Ikuko Kakizaki1, Keiichi Takagaki1,
Yasufumi Endo1, Hitoshi Ikeya2, Teruzo Miyoshi2, Akio Nakane3, Paul H.
Weigel4, and Masahiko Endo1
1Department of Biochemistry and 3Department
of Bacteriology, Hirosaki University School of Medicine, Hirosaki, 2Research
Center, Denki Kagaku Kogyo Co. Ltd., Tokyo, Japan, and 4Department of Biochemistry
and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma
City, OK, USA
Hyaluronan (hyaluronic
acid, HA) is one of the major components of the extracellular matrix, and
is involved in many biological processes of tissue organization, wound
healing, tumor invasion and cancer metastasis, interacting with other extracellular
matrix components. Certain strains of Streptococci are also able to synthesize
HA, and it is not chemically distinguishable from that in mammalian tissues.
Recently, studies on the HA synthesis and its regulation mechanism have
made rapid progress owing to the cloning of the HA synthase genes. We have
previously found that HA synthesis in cultured human skin fibroblasts was
inhibited by 4-methylumbelliferone (MU) with no effect on any other glycosamioglycan
synthesis and a HA deficient extracellular matrix was formed. In order
to elucidate the inhibition mechanism of HA synthesis, we examined the
effect of MU on Streptococcus equi FM100, as a model.
HA synthesis in FM100
cells was also inhibited by treatment with MU1). In this study the effects
of MU on HA synthesis activity and expression level of HA synthase in FM100
cells were examined. When MU was added to the culture medium of FM100 cells,
HA production in the isolated membrane rich fractions was decreased in
a dose-dependent manner. On the contrary, when MU was added to the reaction
mixture in the cell-free HA synthesis experiment, there was no change in
the HA production of the membrane rich fraction. These observations suggested
that MU did not directly inhibit HA synthase activity but finally inhibited
the activity in live cells. Northern and Western blot experiments revealed
that mRNA and protein levels of HA synthase were not affected by treatment
with MU. These data suggested that MU did not inhibit the processes of
transcription and translation of HA synthase.
Thus, it is supposed
that MU may inhibit the posttranslational modification of HA synthase or
inhibit the HA synthesis activity through some regulating factors.
1. Kakizaki, I., Takagaki, K, Endo,
M., Nakane, A., Ikeya, H., and Miyoshi, T. (1999) Abstract in International
Conference on Molecular Interactions of Proteoglycans, Kanagawa,
Japan
P-24
INCREASED LEVELS OF CIRCULATING HYALURONATE IN THE SERA OF PATIENTS WITH
RHEUMATOID ARTHRITIS WITH SPECIAL REFERENCE TO JOINT DESTRUCTION.
Takashi Sawai, Miwa Uzuki
Department of Pathology, Iwate Medical
University School of Medicine
We sought to assess
whether serum levels of hyaluronate(HA) in patients with rheumatoid arthritis
(RA) are elevated and correlated with grade of joint destruction and with
laboratory data. Circulating HA levels were determined by protein binding
assay of serum from 236 patients and 19 healthy controls.
The greatest elevations of HA were
seen in RA patients (350.7+689. ng/ml) than controls (33.7+24.2ng/ml).
Their levels showed only weak correlation to values of CRP in laboratory
data. Although HA levels of sera in stageIII and IV in Steinbrocker's Classification
revealed significantly higher on average than ones in stage I and II (p<0.01),
there were many cases with low HA levels even in stage III and IV. Comparing
HA levels between the cases with progressive and non- progressive type
of hip or knee joint destruction within a year, the former(1143.6+1746.0
ng/ml median 480.1ng/ml) presented significantly higher HA levels than
the latter(245.3+255.8 ng/ml median 140.0ng/ml)(p<0.01). Further, one
of 7 cases followed by 4 years with extremely high levels of HA showed
progression of joint destruction from grade III to IV in Larsen Grade in
bilateral knee joints during these times, though there were no significant
correlations between HA levels and laboratory data.
From above results,
levels of circulating HA in the sera of patients with RA correspond to
the joint process of destruction, not to a result of one.
P-25
HYALURONIC ACID(HA) WITH HIGH MOLECULAR WEIGHT SHOWS HIGH AFFINITY TO
DEGENERATED CARTILAGE IN TISSUE SECTIONS.
Miwa Uzuki, Takashi Sawai
Department of Pathology, Iwate Medical
University, School of Medicine, Morioka, Japan
Hyaluronic acid (HA)
is a major component of synovial fluid and joint cartilage which is deteriorated
in osteoarthritis. Intra-articular injection of HA has been adopted as
conservative therapy. Recently through the development of biotechnological
methods, it has become possible to produce HA with different molecular
weight (MW). In this study we histochemically compared the affinity of
HAs with various sized MWs to tissue specimen, treated with Streptomyces
hyaluronidase. This is considered a model for cartilage deterioration.
HA with four molecular
weights (MWs) were used; HA with a MW of 1.9 x 106 Da., which
was synthesized by Streptococcus equi, and ultrasonically treated
HAs with MWs of 1.5 x 106 , 0.64 x 106 , 0.17 x 106
Da.. Human cartilaginous tissue sections were treated with Streptomyces
hyaluronidase, as a model for cartilage deterioration. These sections were
exposed to the above HA which was produced biotechnologically. The intensity
of HA affinities was compared by three histological methods 1) alcian blue
staining as the classical method 2) FITC-labeled HA, and 3) biotinylated
hyaluronan binding protein (HABP).
1) Alcian Blue In this
study we used four kinds of HA;1.9 x 106 , 1.5 x 106
, 0.64 x 106 , 0.17 x 106 Da.. The group of high
MW, 1.9 x 106 , 1.5 x 106 Da., revealed a higher
affinity to tissue sections comparing to the group with MWs of 0.64
x 106 , 0.17 x 106 Da., although affinity of each
specimen according to different MWs showed no clear difference.
2) FITC In this study,
we used two kinds of FITC-labeled HA; 1.9 x 106 , and 0.8 x
106 Da.. HA with a large MW (1.9 x 106 Da.), showed
a higher affinity to tissue sections than low MW (0.8 x 106
Da.).
3) HABP We examined
the affinity of tissue sections by means of HA of four kinds of MWs; 1.9
x 106 , 1.5 x 106 , 0.64 x 106 , 0.17
x 106 Da.. HA with high molecular weight demonstrated high affinity
to tissue section. Intensity of 3, 3'-diaminobenzidin tetrahydrochloride
(DAB) reaction with biotinylated HABP grew stronger with increasing molecular
weight.
These results revealed
that the affinity of HA to degenerated cartilage increased with increasing
molecular weight.
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