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Post-translational processing of the inter-alpha-trypsin inhibitor in the human hepatoma HepG2 cell line.

ABSTRACT: In human hepatoma HepG2 cells, the serum inter-alpha-trypsin inhibitor (ITI)-like protein is synthesized from two protein precursors, the heavy chain (H) H2 and the light chain (L). Both of them carry sulphate groups involved in the chondroitin sulphate glycosaminoglycan (GAG) linkage, as demonstrated by [35S]sulphate labelling, chondroitinase digestion and inhibition with beta-D-xyloside, an artificial GAG acceptor. While inhibition of N-glycosylation prevented neither the maturation nor the secretion of the ITI-related entities, brefeldin A induced the accumulation of H and L precursors in the cells, therefore blocking subsequent association and maturation of the precursors before their secretion. The enzyme system involved in the ester linkage between H and L chains is localized in the trans-Golgi network since no ITI-like protein could be obtained in the presence of monensin; instead free heavy-chain protein forms and bikunin were secreted in culture supernatants. The ITI-like protein synthesized by HepG2 cells is therefore composed of two heavy chains HC2 linked to two bikunin chains by chondroitin sulphate bridges, although the GAG linkage between HC2 chains is presumably different. Further, a different maturation route leading to restricted heavy-chain forms, Hm and Hd, could be shown.

SUBMITTER: Heron A

PROVIDER: S-EPMC1137266 | biostudies-other | 1994 Sep

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Anticitrullinated protein antibodies (ACPA) and citrullinated proteins play key roles in the pathogenesis of rheumatoid arthritis (RA). Many candidate citrullinated antigens have been identified in joints, but citrullinated proteins in sera are mostly uncertain in patients with RA. We explored the expression of citrullinated proteins in joints and sera of experimental arthritis, and we further investigated their specific expression correlated with the disease activity in patients with RA. METHODS:Citrullinated protein expression in tissues was examined by IHC in peptide glucose-6-phosphate isomerase-induced arthritis (pGIA). Serum citrullinated proteins from pGIA were examined by Western blotting, and the sequence was identified by MS. With the same methods, serum citrullinated proteins were analyzed in patients with RA, primary Sjögren's syndrome, systemic lupus erythematosus, and osteoarthritis as well as in healthy subjects, by Western blotting and MS. In patients with RA, the relationship between the expression of the identified protein (inter-alpha-trypsin inhibitor heavy chain 4 [ITIH4]) and clinical features was evaluated, and the levels of citrullinated ITIH4 were compared before and after biological treatment. The antibody response against citrullinated ITIH4 peptide was measured by enzyme-linked immunosorbent assay. RESULTS:Citrullinated proteins were detected specifically in arthritic joints and sera from pGIA relative to controls. In sera, a common band of citrullinated protein at 120 kDa was revealed, and it fluctuated in parallel with arthritis score of pGIA by Western blotting. Interestingly, in 82% of RA patient sera, similar bands of citrullinated protein were specifically detected. These proteins were identified as citrullinated ITIH4, and especially the R438 site was commonly citrullinated between mice and humans. Citrullinated ITIH4 levels were associated with clinical parameters such as C-reactive protein (CRP), rheumatoid factor, and Disease Activity Score in 28 joints as measured by CRP in patients with RA. Its levels were decreased in correlation with the reduction of disease activity score after effective treatment in patients with RA. Moreover, antibody response to citrullinated epitope in ITIH4 was specifically observed in patients with RA. CONCLUSIONS:Our results suggest that serum citrullinated ITIH4 was specifically increased in patients with RA and could be a novel biomarker for assessing disease activity in patients with RA.

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Post-translational processing of the inter-alpha-trypsin inhibitor in the human hepatoma HepG2 cell line.

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