Human protein HC displays variability in its carboxyl-terminal amino acid sequence

Abstract

1. INTRODUCTION Protein HC (human complex-forming glycoprotein, heterogeneous in charge) is a glycoprotein present in human biological fluids [ 11. It carries yellow- brown chromophores and forms complexes with IgA and albumin to a considerable extent [l]. Although it gives a single band on SDS-polyacryl- amide gel electrophoresis, it shows an extensive charge heterogeneity on isoelectric focussing and on agarose gel electrophoresis which does not decrease after desialylation [ 11. The complete amino acid sequence of protein HC isolated from the urine of an individual was reported in [2]; no evidence for sequence variability of the single polypeptide chain of protein HC was found [3]. This work was under- taken to investigate if the polypeptide chain of protein HC isolated from a pool of urine from several individuals displayed any variability. The polypeptide chain of the protein HC (pool) showed a unique NH2-terminal amino acid sequence up to residue 35, which is identical to the one reported for protein HC (single). However, two different C-ter- minal CNBr fragments were isolated from protein HC (pool). One had the same C-terminal amino acid sequence as reported for protein HC (single) and the other as reported for cut-microglobulin 141, a protein closely related to protein HC. 2. MATERIALS AND METHODS Protein HC was isolated as in [l] from the urine of an individual (J.L.) and from a pool of urine from several individuals. The 2 protein HC preparations are referred to as protein HC (single) and protein HC (pool), respectively. CN7 is the C-terminal CNBr fragment isolated from protein HC (single) as in [3]. Complete reduction and S-carboxy- methylation of the protein preparations was performed in the presence of 6 M guanidinium-HCl [ 11. Hy- drolyses were carried out at 110°C for 20 h using 200 4 5.7 M HCl with 0.02% 2-mercaptoethanol. The analyses were performed using a Beckman 12 l-MB analyzer. Automated amino acid sequence deter- minations by the procedure in [5] were performed on a Beckman 890 C Sequencer using the program in [6]. The phenylthiohydantoin amino acids were identified by high-performance liquid chromato- graphy and in some instances by amino acid analy- sis after back hydrolysis 171. Reduced and [ “C]al- kylated protein HC was dissolved in 70% (v/v) for- mic acid to 20 mg/ml and treated with CNBr with a protein-CNBr ratio of 1:25 (w/w) for 24 h at room temperature [S]. Protein HC (150 pg) or 5 nmol CNBr fragments thereof was used for each of the following carboxypeptidase treatments. Digestion with carboxypeptidase A was carried out in 0.2 M N-methylmorpholine acetate buffer (pH 8.2) for 5 h at 37°C at an enzyme-substrate ratio of 1:lO (w/w) [9]. Digestion with carboxypeptidase B was performed in 0.2 M N-methylmorpholine acetate buffer (pH 8.2) for 16 h at 37°C at an enzyme