Analysis of post‐translational modification of mycobacterial proteins using a cassette expression system

Herrmann, Jean-Louis; Delahay, Robin M.; Gallagher, Alex; Robertson, Brian D.; Young, Douglas B.

doi:10.1016/s0014-5793(00)01553-2

Cited by 50 publications

(67 citation statements)

References 32 publications

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“…The ability to vary protein glycosylation has been described for M. tuberculosis Apa glycoproteins, which show differences in delayed-type hypersensitivity reactions and T-lymphocyte stimulation related to the extent of protein glycosylation (43,44). Other biological roles that carbohydrate modifications on bacterial glycoproteins have been shown to affect include adhesion (45), protection against proteolytic cleavage (46), solubility (47), antigenic variation (48), and protective immunity (49). In C. jejuni, alteration of the N-linked glycosylation pathway by mutation in the pgl locus has already been shown to influence adherence, invasion, colonization, and immunogenicity (3,4).…”

Section: Discussionmentioning

confidence: 99%

Structure of the N-Linked Glycan Present on Multiple Glycoproteins in the Gram-negative Bacterium, Campylobacter jejuni

Young¹,

Brisson

Kelly

et al. 2002

Journal of Biological Chemistry

396

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Section: Discussionmentioning

confidence: 99%

Structure of the N-Linked Glycan Present on Multiple Glycoproteins in the Gram-negative Bacterium, Campylobacter jejuni

Young¹,

Brisson

Kelly

et al. 2002

Journal of Biological Chemistry

396

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“…Several of the immunodominant antigens of M. tuberculosis and M. bovis have been reported to be glycosylated (13,16,32,34). However, to date the unambiguous demonstration of mycobacterial glycosylation has been proved only for the M. tuberculosis antigen MPT32 (16).…”

Section: Discussionmentioning

confidence: 99%

The MPB83 Antigen from Mycobacterium bovis ContainsO-Linked Mannose and (1 → 3)-Mannobiose Moieties

Michell

Whelan

Wheeler

et al. 2003

Journal of Biological Chemistry

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Mycobacterium tuberculosis and Mycobacterium bovis, the causative agents of human and bovine tuberculosis, have been reported to express a range of surface and secreted glycoproteins, although only one of these has been subjected to detailed structural analysis. We describe the use of a genetic system, in conjunction with lectin binding, to characterize the points of attachment of carbohydrate moieties to the polypeptide backbone of a second mycobacterial glycoprotein, antigen MPB83 from M. bovis. Biochemical and structural analysis of the native MPB83 protein and derived peptides demonstrated the presence of 3 mannose units attached to two threonine residues. Mannose residues were joined by a (1 3 3) linkage, in contrast to the (1 3 2) linkage previously observed in antigen MPT32 from M. tuberculosis and the (1 3 2) and (1 3 6) linkages in other mycobacterial glycolipids and polysaccharides. The identification of glycosylated antigens within the M. tuberculosis complex raises the possibility that the carbohydrate moiety of these glycoproteins might be involved in pathogenesis, either by interaction with mannose receptors on host cells, or as targets or modulators of the cell-mediated immune response. Given such a possibility characterization of mycobacterial glycoproteins is a step toward understanding their functional role and elucidating the mechanisms of mycobacterial glycosylation.Protein glycosylation is ubiquitous in eukaryotes and the diverse array of carbohydrate moieties that can be fashioned to a polypeptide backbone makes glycoproteins ideal molecules to allow specific interactions with other molecules. In contrast, the number of reports of this post-translational modification by prokaryotes is comparatively low. Examples of eubacterial glycoproteins identified to date include cell surface or secreted proteins of pathogens that have antigenic properties and play a role in host pathogen interaction (1-6). Glycosylation of pilin has been postulated to enhance the adherence of Neisseria meningitidis to endothelial cells (2), whereas N-glycosylation of the platelet aggregation-associated protein of Staphylococcus sanguis may promote colonization of the endocardium (7, 8).Removal of a sero-specific glycosyl moiety from the flagellin of Campylobacter jejuni has been reported to alter the O antigenicity of the organism (9, 10). Similarly, Romain et al. (11) demonstrated that removal of covalently bound mannose from the Mycobacterium tuberculosis antigen MPT32 reduced by 10-fold its ability to elicit a delayed-type hypersensitivity reaction in guinea pigs immunized with Mycobacterium bovis BCG.M. tuberculosis and M. bovis are closely related members of the "M. tuberculosis complex" (MTb complex) that secrete a series of immunodominant antigens that have been reported to be glycosylated, on the basis of their ability to bind lectins such as concanavalin A (ConA) 1 (12-15). Structural confirmation of protein glycosylation by mycobacteria was provided by detailed chemical compositional analysis of MPT32, a 45...

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“…tuberculosis is indicated by the frequent loss of Nterminal signal sequences during secretion [4, 9 -12, 15] and posttranslational modifications such as lipidation and glycosylation [20,21]. Although rarely observed in eubacteria, in immunology the glycosylated proteins may contribute to the interaction with the mammalian cells during infection [20,21].…”

mentioning

confidence: 99%

“…Although rarely observed in eubacteria, in immunology the glycosylated proteins may contribute to the interaction with the mammalian cells during infection [20,21]. This report utilizes the top-down MS/MS approach [22,23] to identify proteins secreted into the extracellular milieu, in which mixed protein components are ionized and separated directly by Fourier transform MS and then dissociated MS/MS to provide fragment masses for matching against the DNA predicted sequence and for structural characterization of posttranslational modifications.…”

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confidence: 99%

Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry

ElNaggar

Sze

et al. 2003

J. Am. Soc. Mass Spectrom.

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Secreted proteins of Mycobacterium tuberculosis are implicated in its disease pathogenesis and so are considered as potential diagnostic and vaccine candidates. The search for these has been slow, even though the entire genome sequence of M. tuberculosis is now available; of the 620 protein spots resolved by 2-D gel electrophoresis, 114 secreted proteins have been identified, but for only 13 has the primary structure been partly characterized. For comparison, in this top down mass spectrometry (MS) approach the secreted proteins were precipitated from cell culture filtrate, resuspended, and examined directly by electrospray ionization (ESI) Fourier transform MS. The ESI spectra of three precipitates showed 93, 535, and 369 molecular weight (M r ) values, for a total of 689 different values. However, only ϳ10% of these values matched (Ϯ1 Da) the DNA predicted M r values, but these identifications were unreliable. Of nine molecular ions characterized by MS/MS, only one protein match was confirmed, and its isotopic molecular ions were overlapped by those of another protein. MS/MS identified a total of ten proteins by sequence tag search, of which three were unidentified previously. The low success of M r matching was due to unusually extensive posttranslational modifications, including loss of a signal sequence, loss of the N-terminal residue, proteolytic degradation, oxidation, and glycosylation. Although in eubacteria the latter is relatively rare, a 9 kDa protein showed 7 hexose attachments and two 20 kDa proteins each had 20 attachments. For MS/MS, electron capture dissociation was especially effective. (J Am Soc Mass Spectrom 2003, 14, 253-261)

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Analysis of post‐translational modification of mycobacterial proteins using a cassette expression system

Cited by 50 publications

References 32 publications

Structure of the N-Linked Glycan Present on Multiple Glycoproteins in the Gram-negative Bacterium, Campylobacter jejuni

Structure of the N-Linked Glycan Present on Multiple Glycoproteins in the Gram-negative Bacterium, Campylobacter jejuni

The MPB83 Antigen from Mycobacterium bovis ContainsO-Linked Mannose and (1 → 3)-Mannobiose Moieties

Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry

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