Purification and biochemical characterization of the complete structure of a proteolytically modified β‐2‐microglobulin with biological activity

Abstract

A modified form of β‐2‐microglobulin (β‐2‐m) has previously been described to be present in serum from patients suffering from autoimmune diseases, acquiried 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 β‐2‐m from patients with small‐cell lung cancer. Purified urinary β‐2‐m was added to the serum samples incubated at 20°C for five days to obtain a higher yield of modified β‐2‐m (m‐β‐2‐m). m‐β‐2‐m was then purified from serum by gel filtration followed by chromatofocusing of the fractions containing β‐2‐m. m‐β‐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‐β‐2‐m revealed that the protein is missing one lysine residue compared to the composition deduced from the cDNA sequence of β‐2‐m. Amino acid sequence analysis showed that m‐β‐2‐m consists of two polypeptide chains produced by a proteolytic cleavage of β‐2‐m in the disulphide loop. After reduction and alkylation of m‐β‐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 comparision of the amino acid composition of m‐β‐2‐m with the known sequence of β‐2‐m it was possible to deduce the existence of a Ser‐57 in the A chain. Thus proteolytic clevage of β‐2‐m in the intrachain disulphide loop releases the amino acid Lys‐58, which results in a modified form of β‐2‐m with a molecular mass of 11 620 Da as determined by amino acid analysis.