Human prostate-specific antigen: structural and functional similarity with serine proteases.

Abstract

The complete amino acid sequence of the prostate-specific antigen (PA) from human seminal plasma has been determined from analyses of the peptides generated by cyanogen bromide, hydroxylamine, endoproteinases Arg-C and Lys-C. The single polypeptide chain of PA contains 240-amino acid residues and has a calculated Mr of 26,496. An N-linked carbohydrate side chain is predicted at asparagine-45, and O-linked carbohydrate side chains are possibly attached to serine-69, threonine-70, and serine-71. The primary structure of PA shows a high degree of sequence homology with other serine proteases ofthe kallikrein family. The active site residues of histidine, aspartic acid, and serine comprising the chargerelay system of typical serine proteases were found in similar positions in PA (histidine-41, aspartic acid-96, and serine-192). At pH 7.8, PA hydrolyzed insulin A and B chains, recombinant interleukin 2, and-to a lesser extent-gelatin, myoglobin, ovalbumin, and fibrinogen. The cleavage sites of these proteins by PA were chemically analyzed as the a-carboxyl side of some hydrophobic residues, tyrosine, leucine, valine, and phenylalanine, and of basic residues histidine, lysine, and arginine. The chymotrypsin-like activity of PA exhibited with the chromo- (6) and was tested as a marker for postcoital detection in rape investigations (7). Although the clinical utility of PA has been shown, its biological function and chemical structure are not well characterized (8). We report here the complete amino acid sequence of PA and describe the characteristics of its enzymatic properties. MATERIALS AND METHODSPA was purified from human seminal plasma as described (1). PA was finally purified on a large-pore Vydac (Hesperia, CA) C4 column and eluted either with a linear gradient between 70% (vol/vol) buffer A (buffer A = 0.1% trifluoroacetic acid in H20) and 37% (vol/vol) buffer B (buffer B = 0.1% trifluoroacetic acid in acetonitrile) in 280 min or 10% (vol/vol) buffer B and 80% (vol/vol) buffer B in 60 min.The enzymatic activity of PA was assessed on commercially purified proteins including insulin A, insulin B, gelatin, myoglobin, ovalbumin, fibrinogen (Sigma), and recombinant interleukin 2 (Ala-IL2, Cetus). The substrates (1 mg/ml) were dissolved in either 0.1 M ammonium bicarbonate or 50 mM Tris HCl, pH 7.8, containing PA at 0.1 mg/ml. In some cases, a mass ratio (enzyme:substrate) of 1:20 was used. After an 18-hr digestion at 37°C, the peptides were separated by HPLC using a 60-min linear gradient of 0-80% (vol/vol) buffer B. To determine the peptide bond specificity of PA, hydrazinolysis was performed on each ofthe PA/substrate digestion mixtures, substrate alone, and intact PA as described (9). The free carboxyl-terminal amino acids were lyophilized and analyzed on a Beckman 121MB amino acid analyzer.The kinetics ofPA hydrolytic activity on synthetic substrates were studied by monitoring the absorbance change at room temperature using a Hewlett-Packard 8450A spectrophotometer. The following substrates were used: N...

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