Abstract
Human factor I is a two-chain plasma glycoprotein composed of disulfide-linked 50,000- and 38,000-dalton subunits. Analysis of its biosynthesis and postsynthetic processing demonstrated that factor I is synthesized as a single chain precursor (pro-I) that undergoes glycosylation and limited proteolysis to generate the native protein. One of three human hepatoma cell lines, HepG2 , secreted factor I predominantly (70-90%) in a single chain pro-I form. The other cell lines secrete factor I predominantly in its two chain native form. The defect in conversion of pro-I to I in HepG2 was protein specific since other multichain proteins, derived from single chain precursors, the third, fourth, and fifth components of complement were processed normally. Further analysis of the inefficient pro-I to I conversion by HepG2 revealed that Xenopus oocytes injected with HepG2 mRNA secreted factor I in a predominantly two-chain form. In addition, the apparent sizes of native factor I, transferrin, and alpha-1-antitrypsin secreted by the three hepatoma lines differed due to differences in postsynthetic processing.
Highlights
In this report we analyze the biosynthesis of human factor I in three hepatoma-derived cell lines, in acell-free translation system, and in Xenopus oocytes programmed with mRNA from normal humanliver or one of conversion by HepGZ revealed that Xenopus oocytes the human hepatomacell lines (HepG2)
These studies deminjected with HepGZ mRNA secreted factor I in a pre- onstratethat 1) factor I is synthesized as asingle chain dominantly two-chain form
The apparent precursor, pro-I, thatundergoes glycosylation and proteolytic sizes of native factor I, transferrin, and a-l-antitryp- cleavage to generate two-chain native factoI.r2) The HepG2 sin secreted by the three hepatoma lines differed due cell line secretes factor I primarily in the pro-I form while to differences in postsynthetic processing
Summary
Replicate monolayers were pulsed "S-labeled methionine for 1 h. In the presence of tunicamycin, an inhibitorof dolichol phosphatedependent glycosylation (18), the mobility of intracellular factor I was similar to thaotf the primary translation product demonstrated a single precipitin line with whole human plasma and (Fig. 1, lane 3 ). Bovine serum albumin, a rabbit antiserum to factor I or a control antibody (at 1:250 dilution) was incubated with strips for 48 h a t room temperature. The homog- translation system programmed with human liver mRNA ( laneI ) enates were centrifuged in an Eppendorf microfuge for 5min and the and cell lysates of Hep3B cell line culture incubated in the absence supernatants were frozen a t -90 "C.
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