STUDIES OF THE STRUCTURE OF THE METASTASIS-ASSOCIATED 67- KDA LAMININ-BINDING PROTEIN - FATTY-ACID ACYLATION AND EVIDENCE SUPPORTING DIMERIZATION OF THE 32-KDA GENE-PRODUCT TO FORM THE MATURE PROTEIN

The level of expression of the 67 kDa high-affinity laminin binding pr otein (LBP) correlates with the progression of many solid tumors. The cDNA clone for the 67 kDa LBP is sufficient to encode a polypeptide of only 32 kDa, and there is no readily identifiable mechanism for membr ane association. We have overexpressed the transfected 67 kDa hamster LBP in quantities that have enabled us to analyze the membrane-bound f orm of the protein. Treatment of the purified LBP with methyl transest erification reagents, follwed by GC-MS, identified the covalently boun d fatty acids palmitate, stearate, and oleate. The fatty acid modifica tion may provide a mechanism for membrane association. Molecular mass determination by MALDI-TOF MS demonstrated the true molecular mass of the protein to be 66.7 kDa, compatible with the SDS-PAGE observation o f 67 kDa. Treatment of the LBP with neuraminidase, O-glycanase, or End o-F glycosidase has no detectable effect on the apparent molecular mas s of the protein, and the MALDI-TOF MS did not show evidence of mass h eterogeneities typically observed with glycosylated proteins. Reductio n with dithiothreitol or beta-mercaptoethanol had no effect on the app arent molecular mass on SDS-PAGE or on the relative quantities of mole cular mass species on MALDI-TOF MS. The experimentally determined amin o acid composition, however, was found to be consistent with the 67 kD a form being a homodimer of the 32 kDa precursor. Preliminary experime nts also suggest that the high-affinity laminin binding characteristic of the protein may be modulated by an, as yet, unidentified membrane accessory molecule.