Tài liệu Báo cáo khoa học: Experimental proof for a signal peptidase I like activity in Mycoplasma

Experimental proof for a signal peptidase I like activity

in Mycoplasma pneumoniae, but absence of a gene

encoding a conserved bacterial type I SPase

Ina Catrein, Richard Herrmann, Armin Bosserhoff and Thomas Ruppert

Zentrum fu¨r Molekulare Biologie Heidelberg, Universita¨t Heidelberg, Germany

Mycoplasma pneumoniae is a human pathogenic bac￾terium [1,2], characterized by a small genome of 816

kbp [3], the lack of a bacterial cell wall and a parasitic

lifestyle [4].

Some species of the genus mycoplasma, e.g. Myco￾plasma genitalium, Mycoplasma gallisepticum and

Mycoplasma pneumoniae exhibit a flask-like shape,

which is believed to be formed and maintained by a

cytoskeleton-like structure [5–10]. This flask-like shape

is caused by the attachment organelle, an asymmetric

extension of the cell composed of an assembly of

unique proteins [11]. M. pneumoniae interacts with its

host cell by adhering with the attachment organelle to

specific receptors. This interaction takes place only if

the P1 protein, the bacterial main adhesin, is inserted

correctly into the attachment organelle [12]. The

proper insertion depends, among others, on the pro￾teins P40 and P90. Absence of these proteins causes a

random insertion of the P1 protein and a cytadher￾ence-negative phenotype [13]. P1 is encoded by

MPN141 and both, P40 and P90 by MPN142. These

genes are organized, together with MPN140 which

probably encodes a phosphoesterase [14], in the P1

operon [15]. In the original publication these genes

were called ORF4 (MPN140), ORF5 (MPN141) and

ORF6 (MPN142) [15]. The names in brackets are the

gene names according to a recent reannotation [15,16].

MPN142 codes for a protein with a molecular mass of

130 kDa, which, however, has been never identified as

single protein of the expected molecular mass. Instead,

two proteins with molecular masses of about 40 kDa

(P40) and 90 kDa (P90) had been found in SDS ⁄

PAGE and Western blotting experiments [17]. An

enzyme responsible for the processing of the proposed

130-kDa protein has not yet been identified. P40

derives from the N-terminal and P90 from the C-ter￾minal part of the predicted 130-kDa precursor protein.

It was proposed that P40 and P90 are identical with

the proteins B and C missing in certain avirulent

mutants [18,19], but so far this has not been proven

Keywords

chemical assisted fragmentation (CAF);

mass spectrometry; protein modification;

signal peptidase

Correspondence

T. Ruppert, Zentrum fu¨r Molekulare Biologie

Heidelberg, Universita¨t Heidelberg, Im

Neuenheimer Feld 282, 69120 Heidelberg,

Germany

Fax: +49 6221 545891

Tel: +49 6221 546895

E-mail: [email protected]

(Received 15 December 2004, revised

11 March 2005, accepted 7 April 2005)

doi:10.1111/j.1742-4658.2005.04710.x

Although the annotation of the complete genome sequence of Mycoplasma

pneumoniae did not reveal a bacterial type I signal peptidase (SPase I) we

showed experimentally that such an activity must exist in this bacterium, by

determining the N-terminus of the N-terminal gene product P40 of MPN142,

formerly called ORF6 gene. Combining mass spectrometry with a method

for sulfonating specifically the free amino terminal group of proteins, the

cleavage site for a typical signal peptide was located between amino acids 25

and 26 of the P40 precursor protein. The experimental results were in agree￾ment with the cleavage site predicted by computational methods providing

experimental confirmation for these theoretical analyses.

Abbreviations

CID, collision induced fragmentation; SPase I, signal peptidase I.

2892 FEBS Journal 272 (2005) 2892–2900 ª 2005 FEBS