Sequencing of oligoarabinosyl units released from mycobacterial arabinogalactan by endogenous arabinanase: identification of distinctive and novel structural motifs.
The mycobacterial D-arabinofuran is a common constituent of both cell wall mycolyl-arabinogalactan (AG) and the associated lipoarabinomannan (LAM), and is thus accorded critical structural and immunological roles. Despite a well-recognized importance, progress in understanding its full structural characteristics beyond the nonreducing terminal motifs has hitherto been limited by available analytical tools. An endogenous arabinanase activity recently isolated from Mycobacterium smegmatis was previously shown to be capable of releasing large oligoarabinosyl units from AG. Advanced tandem mass spectrometry utilizing both low and high energy collision induced dissociation now afforded a facile way to map and directly sequence the digestion products which were dominated by distinctive Ara18 and Ara19 structural units, together with Ara7 and lesser amount of Ara11 and Ara12. Significantly, evidence was obtained for the first time which validated the linkages and branching pattern of the previously inferred Ara22 structural motif of AG, on which the preferred cleavage sites of the novel arabinanase could be localized. The established linkage-specific MS/MS fragmentation characteristics further led to identification of a galactosamine substituent on the C2 position of a portion of the internal 3,5-branched Ara residue of the AG of Mycobacterium tuberculosis, but not that of the nonpathogenic, fast growing M. smegmatis.
Lee
Arwen
A
Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan.
Wu
Sz-Wei
SW
Scherman
Michael S
MS
Torrelles
Jordi B
JB
Chatterjee
Delphi
D
McNeil
Michael R
MR
Khoo
Kay-Hooi
KH
D020816:Amino Acid Motifs; D001089:Arabinose / Q000737:chemistry; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002473:Cell Wall / Q000201:enzymology / Q000378:metabolism; D040143:Cellulomonas / Q000201:enzymology; D005685:Galactans / Q000737:chemistry* / Q000378:metabolism; D005688:Galactosamine / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011135:Polysaccharides, Bacterial / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
D005685:Galactans; D011134:Polysaccharides; D011135:Polysaccharides, Bacterial; C030080:araban; D005688:Galactosamine; D001089:Arabinose; D006026:Glycoside Hydrolases; C082526:arabinan endo-1,5-alpha-L-arabinosidase; C005653:arabinogalactan