PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF THE COMPLETE STRUCTURE OF A PROTEOLYTICALLY MODIFIED BETA-2-MICROGLOBULIN WITH BIOLOGICAL-ACTIVITY

A modified form of .beta.-2-microglobulin (.beta.-2-m) has previously been described to be present in serum from patients suffering from autoimmune diseases, acquired immune deficiency syndrome and small-cell lung cancer [Plesner, T. and Wiik, A. (1979) Scand. J. Immunol. 9, 247-254; Bhalla et al. (1985) Clin. Chem. 31, 1411-1412; Nissen et al. (1984) Clin. Chim. Acta 141, 41-50]. In the present study we describe the purification and characterization of this modified human serum .beta.-2-m from patients with small-cell lung cancer. Purified urinary .beta.-2-m was added to the serum samples incubated at 20.degree.C for five days to obtain a higher yield of modified .beta.-2-m(m-.beta.-2-m). m-.beta.-2-m was then purified from serum by gel filtration followed by chromatofocusing of the fractions containing .beta.-2-m. m-.beta.-2-m was found to have an apparent molecular mass of 15 kDa and a pI of 5.3 when analyzed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and analytical isoelectric focusing respectively. Amino acid analysis of m-.beta.-2-m revealed that the protein is missing one lysine residue compared to the composition deduced from the cDNA sequence of .beta.-2-m. Amino acid sequence analysis showed that m-.beta.-2-m consists of two polypeptide chains produced by a proteolytic cleavage of .beta.-2-m in the disulphide loop. After reduction and alkylation of m-.beta.-2-m the two chains were separated by reverse-phase high-pressure liquid chromatography. By amino acid sequencing, amino acid residues 1-56 and 59-99 were identified in the A and B chains respectively. By comparison of the amino acid composition of m-.beta.-2-m with the known sequence of .beta.-2-m it was possible to deduce the existence of a Ser-57 in the A chain. Thus proteolytic cleavage of .beta.-2-m in the intrachain disulphide loop releases the amino acid Lys-58, which results in a modified form of .beta.-2-m with a molecular mass of 11620 Da as determined by amino acid analysis.