Abstract
Factor I is a serine proteinase of complement which together with one of several specific cofactors cleaves activation products of the third and fourth components of complement (C3b and C4b) and modulates the activity of C3 convertase. A heterodimer glycoprotein (Mr = 88,000), factor I is synthesized as a single-chain precursor, prepro-I, which undergoes intracellular proteolytic processing. The human hepatoma line HepG2, however, secretes predominantly the single-chain precursor pro-I. In order to determine the molecular basis for this apparent processing defect, factor I cDNA clones were isolated from a HepG2 mRNA-derived library. Sequencing of the largest insert, HI1971, revealed that it contains 14 base pairs of 5' untranslated region, the complete coding sequence for the 583-residue prepro-I (NH2-signal peptide-heavy chain-linking peptide-light chain-COOH), two polyadenylation signals within the 200-base pair 3' untranslated region, and a portion of poly(A) tail. Analysis of the derived protein structure 1) reveals a mosaic multidomain structure of the heavy chain; 2) demonstrates structural similarity between intracellular conversion of pro-I and activation of other serine proteinase zymogens; and 3) indicates that the light chain of factor I resembles most closely the active subunit of tissue plasminogen activator among all serine proteinases and factor D among complement proteinases. Furthermore, this protein sequence was compared to the sequences of factor I cDNA clones isolated from normal human liver libraries and found to be identical. By exclusion, this defines as cellular the basis for the inefficient processing of pro-I by the HepG2 line. Chromosomal localization by the somatic cell hybrid method maps the factor I gene to chromosome 4.
Highlights
From the $Laboratory of Human Biochemistry and rGenetics Division, Children’s Hospital Corporation, Boston, Massachusetts 02115, **Departmentof Hematology-Oncology,Massachusetts General Hospital, Haruard Medical School, Boston, Massachusetts 021 14,and 11Imperial Chemical Industries PLC, Pharmaceuticals Division, Cheshire SKI 0 4TG, United Kingdom
Factor I is a serine proteinase of complement which Factor I (C3b/C4b1inactivator) is a regulatory serine protogether with one of several specific cofactors cleaves teinase of the complement cascade
In order to determine themolecular basis fothr is apparentprocessing defect, factor I cDNA clones were isolated from a HepG2 mRNAderivedlibrary
Summary
Fore, be estimated only for the light (244) but not the heavy chain ((321). Isolation of Factor Z cDNA Clones and Determinationof the The predicted size of pro-I, 63.5 kDa (565 residues), is in Derived mRNA andProtein Structures-Based uponthe good agreement with the estimates for thesize of the nonglyamino acid sequence of the NH,-terminal region of the light cosylated pro-I ( M , = 65,000) and the carbohydrate content chain (lo),' a degenerate 23-mer complementary oligonucle- of 27% (8).The determined amino acid composition of the otide probe was synthesized (Fig. L4). Digestion of the phage DNA withEcoRI permitted assessment of the insert size in the positive clones. The -23-kDa difference betweenthe sizes of unglycosylated and the fully glycosylated pro-I has been determined to be due to N-linked glycosylation (8).
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