The low pH in trans-Golgi triggers autocatalytic cleavage of pre-alpha -inhibitor heavy chain precursor.

Pre-alpha-inhibitor is a plasma protein whose physiological function is still unknown, but in vitro studies suggest that it might be involved in inflammatory reactions. Pre-alpha-inhibitor consists of a 25- and a 75-kDa polypeptide: bikunin and heavy chain 3 (H3), respectively. H3 is synthesized with a 30-kDa C-terminal extension, which is released in the Golgi complex through cleavage between an Asp and a Pro residue. We now provide evidence that this cleavage is triggered by the low pH in the late Golgi and occurs through an intramolecular process. First, incubation in vitro of the H3 precursor (proH3) at pH 6.0 or lower results in rapid cleavage of the protein. Second, the rate of the cleavage reaction does not depend on the concentration of proH3 and is not affected by the presence of various protease inhibitors. Third, raising the pH in organelles of cells producing proH3 abolishes cleavage during secretion. The amino acid residues near the cleavage site of proH3 differ from those of previously described self-cleaving proteins, indicating that the mechanisms of scission are different.

Binding of Zn(2+) to the plasma protein inter-alpha-inhibitor.

Inter-alpha-inhibitor (IalphaI) is a serum protein consisting of a chondroitin-sulfate-containing protein of 25 kDa (bikunin) and two other polypeptides of 75-80 kDa (heavy chains 1 and 2). The physiological function of IalphaI is unclear but recent results suggest that it is required for the formation of the extracellular matrix of certain cell types and that it has anti-inflammatory activity. It was previously reported that IalphaI isolated from serum contains bound Zn(2+), but details of this binding are lacking. Using equilibrium dialysis, we have found that when the free Zn(2+) concentration is raised from 0.3 to 50 micromol/L, the number of bound ions increases from 0.1 to 7. The concentration of free Zn(2+) in plasma is in the nanomolar range; our results therefore suggest that inter-alpha-inhibitor does not contain stoichiometric amounts of zinc ions under normal in vivo conditions.

Intracellular proteolytic processing of the heavy chain of rat pre-alpha-inhibitor. The COOH-terminal propeptide is required for coupling to bikunin.

Pre-alpha-inhibitor is a serum protein consisting of two polypeptides named bikunin and heavy chain 3 (H3). Both polypeptides are synthesized in hepatocytes and while passing through the Golgi complex, bikunin, which carries a chondroitin sulfate chain, becomes covalently linked to the COOH-terminal amino acid residue of H3 via its polysaccharide. Immediately prior to this reaction, a COOH-terminal propeptide of 33 kDa is cleaved off from the heavy chain. Using COS-1 cells transfected with rat H3, we found that in the absence of bikunin, the cleaved propeptide remained bound to the heavy chain and that H3 lacking the propeptide sequence did not become linked to coexpressed bikunin. Sequencing of H3 secreted from COS-1 cells showed that part of the molecules had a 12-amino acid residue long NH2-terminal propeptide. Cleavage of this propeptide, which occurred in the endoplasmic reticulum, was found to require basic amino acid residues at P1, P2, and P6 suggesting that it is mediated by a Golgi enzyme in transit. Deletion of the NH2-terminal propeptide or blocking of its release affected neither transport nor coupling of the heavy chain to bikunin.

Intracellular coupling of bikunin and the heavy chain of rat pre-alpha-inhibitor in COS-1 cells.

Pre-alpha-inhibitor is a serum protein consisting of two polypeptides: bikunin of 16 kDa, which carries an 8 kDa chondroitin sulphate chain, and heavy chain 3 (H3) of 74 kDa. The two polypeptides are linked through an ester bond between an internal N-acetylgalactosamine residue of the chondroitin sulphate chain and the C-terminal aspartic acid residue of H3. Both bikunin and H3 are synthesized by hepatocytes and become linked as they pass through the Golgi complex. H3 is synthesized with both N- and C-terminal extensions which are released during intracellular transport. To be able to analyse the assembly of pre-alpha-inhibitor in detail, we have cloned and sequenced the cDNA of rat H3. Upon expression of the protein in COS-1 cells, both propeptides were found to be released. Furthermore, co-expression of H3 and bikunin resulted in the two polypeptides becoming coupled, indicating that cells other than hepatocytes may have the capacity to form chondroitin sulphate-containing links.

iyk, a novel intracellular protein tyrosine kinase differentially expressed in the mouse mammary gland and intestine.

Using a polymerase chain reaction based cloning strategy and conventional cDNA library screening we have characterized a novel intracellular protein tyrosine kinase from mouse mammary tissue. The gene encodes a protein with structural characteristics reminiscent of the src family of protein tyrosine kinases, a partial myristylation signal with glycine in position +2, SH3 and SH2 domains followed by a C-terminal tyrosine kinase domain. Interestingly, a potential bipartite nuclear localizing signal is located within the SH2 domain. A data base search revealed the highest homology to the human frk/rak gene. Northern blot analysis of mammary gland development indicated preferential epithelial expression in resting mammary glands and up-regulation in mammary tumours. Expression in organs was highest in the intestinal tract and was confined to the small intestine. We have named this gene iyk, (intestine tyrosine kinase), reflecting its pattern of expression.