Post-translational processing of the inter-α-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.

Involvement of the three inter‐α‐trypsin inhibitor (ITI) heavy chains in each member of the serum ITI family

“…Indeed, the observed differences in the respective serum concentrations of ITI family members may result from a balance between IT/synthesis and metabolic pathways with a low stability of particular components, as demonstrated, for instance, with human leukocyte elastase [33]. In this respect, it is worth noting that HepG2 cells do not synthesise two-chain structures which may only be found after partial digestion of the ITI molecule with chondroitinase [32] although it has been proposed that interleukin 6 induces in HepG2 cells the synthesis of a Pal form whose composition has not been ascertained [34].…”

“…Indeed, several elements challenge the commonly accepted composition of ITI, viz. only two heavy chains (HC1 and HC2) + bikunin: (1) in foetal calf serum, ITI is devoid of the HC1 chain and composed of HC2, HC3 and bikunin chains as herein described in agreement with the protein-sequencing results [29]; (2) the H3 chain is found in human serum ITI, although in very low amounts, thus explaining why it had not been previously detected by protein-sequencing; (3) acute-phase mediators induce variations in the transcription rates of ITI genes, including opposite effects on ITI H 1 and H2 genes [30]; (4) Wisniewski et al [31] recently purified a serum protein with high affinity for TSG-6, a member of the hyaladherin family; this protein was shown to be an ITI molecule which only displayed H2 and bikunin chains by protein-se-quencing; furthermore, the same component was found in supernatants of HepG2 cells, which in fact synthesise an IT/form only composed of HC2 and bikunin chains [32]. We, therefore, propose a multichain structure for serum ITI involving two identical or two different heavy chains, with a major involvement of ill and H2.…”

“…They allowed a better understanding of ITI synthesis and maturation in cellular models [32,35] and moreover permitted a very simple analysis of all ITI family members in complex mixtures, thereby suggesting alternatives to their structuring. However, the mechanism responsible for the coexistence of ITI and HC/bikunin forms in serum, i.e.…”

Proinflammatory cytokines up-regulate synthesis and secretion of urinary trypsin inhibitor in human hepatoma HepG2 cells

“…In hepatocytes, different types of UTI-containing proteins are formed by assembly of UTI with one or two of the three evolutionary related heavy chains (H1, H2, and H3); these proteins comprise inter-␣-inhibitor (I␣I) family members, including I␣I, pre-␣-inhibitor (p␣I), inter-␣-like inhibitor (I␣LI) and free UTI. I␣I, p␣I, and I␣LI are composed of H1+H2+UTI, H3+UTI, and H2+UTI, respectively [4,5]. During inflammation, UTI is cleaved from I␣I family proteins by limited proteolysis in the peripheral circulation or at the inflammation site [6][7][8].…”

Urinary Trypsin Inhibitor, an Alternative Therapeutic Option for Inflammatory Disorders

“…I I, p I, and I LI are composed of HC1 + HC2 + UTI, HC3 + UTI, and HC2 + UTI, respectively [5,6]. Its specific activity was 2,613 U/mg protein, one unit being the amount necessary to inhibit the activity of 2μg trypsin (3,200 NFU/mg, Canada Packers) by 50% [7].…”