MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne's Disease

Bannantine, John P.; Lingle, Cari; Stabel, Judith R.; Ramyar, Kasra X.; Garcia, Brandon L.; Raeber, Alex J.; Schacher, Pascal; Kapur, Vivek; Geisbrecht, Brian V.

doi:10.1128/cvi.00195-12

Cited by 14 publications

(18 citation statements)

References 35 publications

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“…Based on BLAST analysis of the NCBI sequence database combined with experimental data, 19 we suggest that the MAP_1272c protein is 279 amino acids in length (29.2 kDa) and not 316 as annotated in the K-10 genome sequence. 17 MAP_1204 encodes a 244 amino acid protein with a calculated molecular weight of 25.4 kDa.…”

Section: Characteristics Of Map_1204 and Map_1272cmentioning

confidence: 99%

“…Two monoclonal antibodies were also developed against MAP_1272c and their binding sites and biochemical properties have been determined. 19 We have now extended those studies by examining the structure and catalytic properties of these proteins in MAP. Here we demonstrate that while both proteins display a conserved substrate-binding groove, one NlpC/P60 domain-containing protein hydrolyzes peptidoglycan while a second one is nonhydrolytic.…”

Section: Introductionmentioning

confidence: 94%

“…19,37 In addition to those MBP fusion proteins, truncated versions of these genes were fused to a polyhistidine tag and Tobacco Etch Virus (TEV) recognition site and subcloned into the E. coli expression vector pT7HMT using methods previously described. 40 A series of His-tagged MAP_1272c truncations have been described in detail elsewhere 19 ; however, this article made extensive use of only a form of MAP_1272c consisting of residues Ala27 to Thr 168 . Similarly, MAP_1204 was truncated such that only the 136 residues from Val 109 to Tyr 244 were expressed.…”

Section: Protein Production and Purificationmentioning

confidence: 99%

“…Therefore, we adopted the convention used in our previous study that set Met38 as Met1, making the full-length MAP_1272c protein from 1 to 279 amino acids and the first 25 amino acids comprising the signal sequence. 19 …”

Section: Protein Production and Purificationmentioning

confidence: 99%

“…Although both MAP_1204 and MAP_1272c proteins are immunogenic, MAP_1272c in particular has been observed as a strong antigen in multiple studies. [19][20][21] Our group has also evaluated MAP_1204 and MAP_1272c as part of a subunit vaccine cocktail against Johne's disease in mice. 21 Those results showed a significant decrease in MAP colonization of the vaccinated mouse intestine.…”

Section: Introductionmentioning

confidence: 99%

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NlpC/P60 domain‐containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan

et al. 2016

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A subset of proteins containing NlpC/P60 domains are bacterial peptidoglycan hydrolases that cleave noncanonical peptide linkages and contribute to cell wall remodeling as well as cell separation during late stages of division. Some of these proteins have been shown to cleave peptidoglycan in Mycobacterium tuberculosis and play a role in Mycobacterium marinum virulence of zebra fish; however, there are still significant knowledge gaps concerning the molecular function of these proteins in Mycobacterium avium subspecies paratuberculosis (MAP). The MAP genome sequence encodes five NlpC/P60 domain-containing proteins. We describe atomic resolution crystal structures of two such MAP proteins, MAP_1272c and MAP_1204. These crystal structures, combined with functional assays to measure peptidoglycan cleavage activity, led to the observation that MAP_1272c does not have a functional catalytic core for peptidoglycan hydrolysis. Furthermore, the structure and sequence of MAP_1272c demonstrate that the catalytic residues normally required for hydrolysis are absent, and the protein does not bind peptidoglycan as efficiently as MAP_1204. While the NlpC/P60 catalytic triad is present in MAP_1204, changing the catalytic cysteine-155 residue to a serine significantly diminished catalytic activity, but did not affect binding to peptidoglycan. Collectively, these findings suggest a broader functional repertoire for NlpC/P60 domain-containing proteins than simply hydrolases. Keywords: Mycobacterium; Johne's disease; crystal structure; peptidoglycan; proteins; antigens; paratuberculosis Additional Supporting Information may be found in the online version of this article.Importance: Johne's disease in ruminant livestock is caused by the bacterium Mycobacterium avium subspecies paratuberculosis. This research describes the functional aspects of two proteins that show promise in a subunit vaccine for Johne's disease. Through crystal structure determination and amino acid modification, we demonstrate that although both proteins have a similar structure, one of them lacked hydrolytic activity on peptidoglycan. We show that a specific amino acid is likely responsible for this lack of hydrolytic activity.

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Section: Characteristics Of Map_1204 and Map_1272cmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 94%