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Chemical analysis of processing of spiralin, the major lipoprotein of Spiroplasma melliferum

Le Hénaff, Michel, Fontenelle, Catherine
Archives of microbiology 2000 v.173 no.5-6 pp. 339-345
Spiroplasma melliferum, acylation, biogenesis, carbon, chemical analysis, cysteine, lipoproteins, membrane proteins, methionine, myristic acid, palmitic acid, plasma membrane, polyacrylamide gel electrophoresis, signal peptide, stearic acid
The plasma membrane of Spiroplasma melliferum contains a major membrane-associated lipoprotein called spiralin. In this study, the processing pathway of spiralin was investigated by chemical analysis of the purified protein and by using [³⁵S]cysteine, [³⁵S]methionine, [¹⁴C]myristic acid (¹⁴C-14:0), [¹⁴C]palmitic acid (¹⁴C-16:0), and globomycin. SDS-PAGE analysis of membrane proteins showed the leader peptide cleavage of prospiralin and provided evidence for an apparent selectivity in the acylation: the unprocessed protein was labelled with ¹⁴C-16:0 only (O-ester-linked acyl chains), and the mature form with both ¹⁴C-labelled fatty acids (O-ester-linked + amide-linked chains). Chemical analysis of the purified protein revealed that spiralin contains S-glycerylcysteine and is covalently modified with two O-ester-linked acyl chains and one amide-linked fatty acid chain. However, a specific selectivity in the O- and the N-acylations was not confirmed; palmitate and stearate were the major components. The amounts of O-ester- and amide-linked acyl chains, the resistance to Edman degradation and the presence of S-glycerylcysteine together indicate that spiralin is a "classical" lipoprotein (i.e. is triacylated) and is probably processed by a mechanism similar to that described for gram-negative eubacteria. On the basis of these findings, a biogenesis pathway for spiralin is proposed.