Abstract
Pre-alpha-inhibitor is a serum protein consisting of two polypeptides, the heavy chain and bikunin, covalently linked through an ester bond between the chondroitin sulfate chain of bikunin and the alpha-carboxyl group of the carboxyl-terminal residue of the heavy chain. The heavy chain is synthesized with a carboxyl-terminal extension, which is cleaved off just before the link to bikunin is formed. Our earlier studies indicate that this extension mediates the cleavage, and we have now found that a short segment on the amino-terminal side of the cleavage site is also required for the reaction. Furthermore, we previously showed that coexpression of the heavy chain precursor and bikunin in COS-1 cells leads to linkage, and we have now used this system to identify a His residue in the carboxyl-terminal extension that is specifically required for the intracellular coupling of the two proteins. In addition, we have shown that another chondroitin sulfate-containing protein, decorin, will also form a complex with the heavy chain, as will free chondroitin sulfate chains. These results suggest that in vivo there might be other, as yet unknown, chondroitin sulfate-containing polypeptides linked to the heavy chain.
Highlights
Bikunin (Bk)1 is a 25-kDa chondroitin sulfate-containing protein consisting of two Kunitz-type protease inhibitor domains, which is secreted by hepatocytes
We previously showed that coexpression of the heavy chain precursor and bikunin in COS-1 cells leads to linkage, and we have used this system to identify a His residue in the carboxyl-terminal extension that is required for the intracellular coupling of the two proteins
The heavy chains are synthesized with a carboxyl-terminal extension (CTX), which is cleaved off when the proteins reach the Golgi complex [13, 14]
Summary
Bikunin; P␣I, pre-␣-inhibitor; H3, heavy chain 3; pH3, heavy chain 3 precursor; CTX, carboxyl-terminal extension of heavy chain 3; CS, chondroitin sulfate; DS, dermatan sulfate; pNPXP, p-nitrophenyl--D-xylopyranoside. This process is not known, but in vitro experiments have shown that hyaluronan molecules bearing heavy chains are less sensitive to degradation by free radicals [8]. The heavy chains are synthesized with a carboxyl-terminal extension (CTX), which is cleaved off when the proteins reach the Golgi complex [13, 14] Both bikunin and the heavy chains are synthesized in hepatocytes, and immediately after the cleavage of the CTX, they become linked through an ester bond between the chondroitin sulfate chain of bikunin and the ␣-carboxyl group of the carboxyl-terminal amino acid residue of the heavy chains [4, 15] (Fig. 1). Because bikunin is the most abundant CS-containing proteoglycan produced by hepatocytes and so far the only protein known to be covalently linked to the heavy chains, our results suggest that there might be other, minor chondroitin sulfatecontaining proteins linked to heavy chains in vivo
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