Human pre‐α‐trypsin inhibitor‐precursor heavy chain cDNA and deduced amino‐acid sequence

Abstract

Pre-a-trypsin inhibitor (Pal) is a serine-proteinase inhibitor of M, 130000 found in human serum. This protein belongs to the family of proteins called inter-a-trypsin inhibitor (ITI). Pa1 is composed of a heavy chain (HC3) and of a light chain (bikunin), synthesized by two separate mRNA. Bikunin is identical to the IT1 light chain, the structure of which has already been established. The HC3 is obtained from a precursor called H3. The bikunin is covalently linked to HC3 by a chondroitin-4-sulfate glycosaminoglycan. We report here the H3 full-length cDNA sequence and the deduced amino-acid sequence of the heavy-chain H3 precursor. The high degree of similarity between the nucleotide and amino-acid sequences of IT1 heavy-chain families H1, H2, H3 is examined with respect to their probable structure and assembly with bikunin in the final proteins, Pal and ITI.Pre-a-trypsin inhibitor (PaI) is a recently described glycoprotein of M, 130000 [l], structurally related to the intera-trypsin-inhibitor (ITI) family of serine-proteinase inhibitors. Pal is composed of one heavy-chain of M, 90000, called HC3, and one light chain of M , 30000 named bikunin because it is composed of two pancreatic-trypsin-inhibitor(Kunitz)-type domains [2]. The isolation and sequence of ITI-light-chain cDNA [3, 41 and gene [5] showed that they code for two tandemly arranged proteins : a-1-microglobulin and bikunin. Pal chains are covalently linked to each other by an unusual structure among plasma proteins: the interchain linkage is mediated through esterification of the a-carbon of the C-terminal Asp of HC3 with the C6 of an internal N-acetylgalactosamine of the glycosaminoglycan chain 0-linked to SerlO of the bikunin [6]. The sequence analysis of peptides derived from the proteolysis of Pal indicates that the heavy-chain precursor (heavy-chain precursors are called H, whereas mature heavy chains are denominated HC, according to [l]) of this protein is coded by the IT1 H3 heavy-chain cDNA which we previously described [7]. We have also shown that the H3 gene is located in the p211-p212 region of chromosome 3 [7]. Abbreviations. PaI, pre-a-trypsin inhibitor; ITI, inter-a-trypsin inhibitor; H1, H2 and H3, heavy chain precursor of inter-a-trypsin inhibitor family; HC1, HC2 and HC3, mature heavy chain of inter-atrypsin-inhibitor family; RACE, rapid amplification of cDNA ends.Enzymes. Reverse transcriptase (EC 2.7.7.49) ; DNA polymerase I, Tuq DNA polymerase (EC 2.7.7.7).Note. The novel nucleotide sequence data reported here have been deposited in the EMBL/GenBank library under accession number X 67055.In the present study, we report the complete cDNA sequence of H3 heavy chain, and therefore the complete amino-acid sequence of Pa1 precursor HC3 heavy chain. The amino-acid-sequence similarity between ITI-heavy-chain families H1, H2 and H3 was also studied. MATERIALS AND METHODS ReagentsRestriction endonucleases, reverse transcriptase, M13 mp18, M13 mp19 and pUC 18 vectors were from Bethesda Research Laboratories. Nitrocellulose filters (BA 85) we...

The Low pH in Trans-Golgi Triggers Autocatalytic Cleavage of Pre-α-inhibitor Heavy Chain Precursor

“…Earlier studies have shown that the heavy chains of pre-and inter-␣-inhibitor are synthesized as precursors with C-terminal extensions, which are cleaved off during transport to the cell surface; for all of these polypeptides cleavage seems to occur between an Asp and a Pro residue (15,16). In the present study we found that incubation at low pH of the precursor of pre-␣-inhibitor heavy chain (proH3) induced cleavage of its C-terminal extension.…”

“…The heavy chains are synthesized as precursors with Cterminal extensions of about 30 kDa (15)(16)(17). These fragments are released in the Golgi complex by cleavage between an Asp and a Pro residue (9).…”

Structural Characterization of Inter-α-inhibitor

“…4A and 5A) is in agreement with this conclusion. Furthermore, there is a high frequency of the structure-breaking amino acid proline near the C termini of H1 and H2 (39). The fact that the proteinase-sensitive parts of the heavy chains constitute approximately half the polypeptides (see Fig.…”

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

“…The peptide sequence of the light chain, deduced from that of the cDNA [5,6], corresponds to the precursor of two tandemly arranged proteins, ~l-microglobulin and bikunin (responsible for the protease inhibitory activity), which separate at the time of ITI maturation [7,8]. Three mRNAs coding for distinct heavy-chain peptides, called H1, H2 and H3, were then described [9][10][11]; they showed highly similar amino acid sequences. Further, the ITI-related proteins are synthesized in the liver by four genes located on three different chromosomes [12].…”

“…As MBP is a strictly bacterial protein, the anti-MBP activity of these new antisera was unlikely to restrict in any way their use for the analysis of human products and this turned out to be true. Despite the high sequence similarity of ITI heavy chains [11], the anti-MBP-H antisera did not show any cross-reaction between them (Fig. 1).…”