Proteins containing convalenty linked carbohydrate play vital roles in the biology of cells, being abundantly represented among secretory products as well as cell surface and basement membrane constituents. The carbohydrate units, with their great potential for diversity, have been implicated in recognition phenomena involving the interaction of cells with each other as well as with substrata and circulating molecules and therefore have relevance to disease processes ranging from neoplasia to diabetes. It is the objective of this research proposal to continue our studies on the structure of the glycoprotein components of cell surfaces and basement membranes, to evaluate their biological role in defined model systems and to pursue our exploration of the biochemical basis of the diabetic renal filtration defect. Furthermore, we will investigate the structure of the sulfated carbohydrate units which we recently detected in thyroglobulins. The carbohydrate chains of glomerular basement membrane and lens capsule components, including heparan sulfate proteoglycan, entactin and type IV collagen, will be defined. In regard to the proteoglycan, particular attention will be given to the distribution of sulfate groups, nature of the core protein and antigenicity as well as binding reactivities of various regions of the molecule; moreover an assessment of anticoagulant activity of the heparan sulfate chains will be made and structural alterations in the diabetic state will be evaluated. Cell-basement membrane interactions will be studied in our lens capsule model system and a search for a type IV collagen receptor on lens cells will be made; the role of the Glc-Gal-Hyl units of collagen in the binding of cells to basement membranes will be evaluated. The disposition of macromolecular components will be probed with immunochemical procedures from both faces of the lens capsule and this basement membrane will also be used to study the migration of polymorphonuclear leukocytes through basement membranes. Thyroid cells will be employed for the study of cell surface glycoprotein structure and cell-cell interactions. Included in these investigations will be the thyrotropin receptor and GP-3, a major carbohydrate rich component. The effect of polarity inversion on the surface distribution of glycoproteins will be evaluated and the role of glycoconjugates on interactions leading to follicle formation will be explored. Studies on the sulfated N-linked carbohydrate units of calf and human thyroglobulins should extend our understanding of this prohormone's structure.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK017325-17
Application #
3225720
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1976-06-01
Project End
1991-05-31
Budget Start
1989-06-01
Budget End
1990-05-31
Support Year
17
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Joslin Diabetes Center
Department
Type
DUNS #
071723084
City
Boston
State
MA
Country
United States
Zip Code
02215
Spiro, Robert G (2002) Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology 12:43R-56R
Edge, A S; Spiro, R G (2000) A specific structural alteration in the heparan sulphate of human glomerular basement membrane in diabetes. Diabetologia 43:1056-9
Spiro, R G; Bhoyroo, V D (1998) Characterization of a spleen sulphotransferase responsible for the 6-O-sulphation of the galactose residue in sialyl-N-acetyl-lactosamine sequences. Biochem J 331 ( Pt 1):265-71
Chandra, N C; Spiro, M J; Spiro, R G (1998) Identification of a glycoprotein from rat liver mitochondrial inner membrane and demonstration of its origin in the endoplasmic reticulum. J Biol Chem 273:19715-21
Karaivanova, V K; Spiro, R G (1998) Sulphation of N-linked oligosaccharides of vesicular stomatitis and influenza virus envelope glycoproteins: host cell specificity, subcellular localization and identification of substituted saccharides. Biochem J 329 ( Pt 3):511-8
Karaivanova, V K; Luan, P; Spiro, R G (1998) Processing of viral envelope glycoprotein by the endomannosidase pathway: evaluation of host cell specificity. Glycobiology 8:725-30
Edge, A S; Spiro, R G (1997) Structure of the O-linked oligosaccharides from a major thyroid cell surface glycoprotein. Arch Biochem Biophys 343:73-80
Spiro, R G; Zhu, Q; Bhoyroo, V et al. (1996) Definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver Golgi. J Biol Chem 271:11588-94
Shen, G Q; Kresbach, G; Spiro, M J et al. (1995) Evaluation of the cell specificity and sulfate dependence of glomerular extracellular matrix proteoglycan synthesis. Arch Biochem Biophys 321:83-93
Spiro, M J; Spiro, R G (1992) Monosaccharide determination of glycoconjugates by reverse-phase high-performance liquid chromatography of their phenylthiocarbamyl derivatives. Anal Biochem 204:152-7

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