Distinct steps in the biosynthetic pathway of glycoproteins have been defined. However the mechanism, regulation and biological significance of many of them are still unclear. We propose to study the details of proteolytic processing of the single chain precursor of native C3b/C4b inactivator (factor I), a two chain serine protease. This will be possible because of the availability of three hepatoma cell lines, one of which, HepG2, secretes factor I in a predominantly (70-90%) single chain, pro-I form. In contrast injection of HepG2 mRNA into Xenopus oocytes results in secretion of predominantly two chain native factor I. These data indicate a defect in precursor protein structure or in post-synthetic processing. The proposed studies will determine the nature of the defect and therefore should lead to better understanding of post-synthetic processing. We will undertake sequence analysis of the pro-I signal peptide, deterine the subunit order in pro-I, perform amino acid analysis of the cleavage region of factor I produced by normal and mutant cell lines and in the Xenopus oocyte system as well as similar analysis of in vitro cleavage factor I. This will be accomplished by metabolic labelling, immunoprecipitation, SDS-PAGE, microradiosequencing and in vitro proteolysis. In addition, we will analyze the effects of differential glycosylation of factor I, transferrin and alpha -1- antitrypsin on their biosynthesis in the three hepatoma cell lines and in Xenopus oocytes. This will be done by metabolic labelling, deglycosylation (TFMSA, Endo-H, and neuraminidase), glycosylation inhibition (tunicamycin, swainsonine and monensin), immunoprecipitation and SDS PAGE.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM033293-03S1
Application #
3282809
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1984-04-01
Project End
1988-01-19
Budget Start
1987-08-01
Budget End
1988-01-19
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
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