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

Author:
Le Hénaff, Michel, Fontenelle, Catherine
Source:
Archives of microbiology 2000 v.173 no.5-6 pp. 339-345
ISSN:
0302-8933
Subject:
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
Abstract:
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.
Agid:
1210521