ProGlyProt ID BC153 Organism Information Organism Name Mycobacterium tuberculosis H37Rv Domain Bacteria Classification Family: Mycobacteriaceae Suborder: Corynebacterineae Order: Actinomycetales Subclass: Actinobacteridae Class: Actinobacteria Division or phylum: "Actinobacteria" Taxonomic ID (NCBI) 1773 Genome Sequence (s) GeneBank BX842578.1 EMBL BX842578 Gene Information Gene Name Apa (Rv1860) or modD NCBI Gene ID 885896 GenBank Gene Sequence 885896 Protein Information Protein Name Alanine and proline-rich secreted protein Apa (50/ UniProtKB/SwissProt ID Q50906 NCBI RefSeq YP_177849.1 EMBL-CDS CAE55438.1 UniProtKB Sequence >sp|Q50906|APA_MYCTU Alanine and proline-rich secreted protein apa OS=Mycobacterium tuberculosis GN=apa PE=1 SV=1 MHQVDPNLTRRKGRLAALAIAAMASASLVTVAVPATANADPEPAPPVPTTAASPPSTAAA PPAPATPVAPPPPAAANTPNAQPGDPNAAPPPADPNAPPPPVIAPNAPQPVRIDNPVGGF SFALPAGWVESDAAHFDYGSALLSKTTGDPPFPGQPPPVANDTRIVLGRLDQKLYASAEA TDSKAAARLGSDMGEFYMPYPGTRINQETVSLDANGVSGSASYYEVKFSDPSKPNGQIWT GVIGSPAANAPDAGPPQRWFVVWLGTANNPVDKGAAKALAESIRPLVAPPPAPAPAPAEP APAPAPAGEVAPTPTTPTPQRTLPA Sequence length 325 AA Subcellular Location Secreted Function Immunodominant antigen. Elicits potent DTH (delayed-type hypersensitivity) responses in living-BCG-immunized guinea pigs..Stimulation of peripheral blood mononuclear cells from PPD positive individuals with Apa induces a Th1 type lymphoproliferative response with expansion of both CD4+ and CD8+ cells and increased IFN-γ production. Protein Structure PDB ID Glycosylation Status Glycosylation Type O (Thr) linked Experimentally Validated Glycosite(s) in Full Length Protein (Signal peptide: 1-39) T49, T57, T66, T316 Experimentally Validated Glycosite(s ) in Mature Protein T10, T18, T27, T277 Glycosite(s) Annotated Protein Sequence >sp|Q50906|APA_MYCTU Alanine and proline-rich secreted protein apa OS=Mycobacterium tuberculosis GN=apa PE=1 SV=1 MHQVDPNLTRRKGRLAALAIAAMASASLVTVAVPATANADPEPAPPVPT*(49)TAASPPST*(57)AAA PPAPAT*(66)PVAPPPPAAANTPNAQPGDPNAAPPPADPNAPPPPVIAPNAPQPVRIDNPVGGF SFALPAGWVESDAAHFDYGSALLSKTTGDPPFPGQPPPVANDTRIVLGRLDQKLYASAEA TDSKAAARLGSDMGEFYMPYPGTRINQETVSLDANGVSGSASYYEVKFSDPSKPNGQIWT GVIGSPAANAPDAGPPQRWFVVWLGTANNPVDKGAAKALAESIRPLVAPPPAPAPAPAEP APAPAPAGEVAPTPTT*(316)PTPQRTLPA Sequence Around Glycosites (21 AA) ADPEPAPPVPTTAASPPSTAA VPTTAASPPSTAAAPPAPATP STAAAPPAPATPVAPPPPAAA PAGEVAPTPTTPTPQRTLPA Glycosite Sequence Logo seqlogo Glycosite Sequence Logo Technique(s) used for Glycosylation Detection Schiff's staining, peroxidase-conjugated concanavalin A (ConA)-binding Technique(s) used for Glycosylated Residue(s) Detection N-terminal amino acid sequencing coupled with fast atom bombardment-mass spectrometry (FAB-MS) Protein Glycosylation- Implication The presence of the mannose residues on the Apa protein was essential for the antigenicity of the molecules in T-cell-dependent immune responses in vitro (proliferation assay) and in vivo (delayed type hypersensitivity or DTH reaction). The deglycosylated antigen was 10-fold less active than native molecules in eliciting DTH. Mannosylation of antigen leads to selective targeting and subsequent greater presentation by dendritic cells. Glycan Information Glycan Annotation Linkage: αMan-Thr. 34.6% sugar detected. α-D-Manp(1→2)α-D-Manp (mannobiose), α-D-Manp (single mannose), α-D-Manp(1→2)α-D-Manp(1→2)α-D-Manp (mannotriose) are present. T10 and T18 are glycosylated with mannobiose, T27 with single mannose, and T277 with either of the three. The majority of the antigen species bear six, seven, or eight mannose residues (22, 24, and 17%, respectively), while others three, four, or five mannoses (5, 9, and 14%, respectively). Technique(s) used for Glycan Identification GC-MS of partially methylated alditol acetates obtained by trifluoro acetic acid (TFA) hydrolysis of permethylated oligoglycosyl alditols which are released by β-elimination of the glycopeptides. Protein Glycosylation linked (PGL) gene(s) OST Gene Name pmt/rv1002c OST NCBI Gene ID 887882 OST GenBank Gene Sequence 887882 OST Protein Name Protein O mannosyltransferase OST UniProtKB/ SwissProt ID O05586 OST NCBI RefSeq NP_215518.1 OST EMBL-CDS CAB08157.1 OST UniProtKB Sequence >sp|O05586|Y1002_MYCTU Uncharacterized protein Rv1002c/MT1031 OS=Mycobacterium tuberculosis GN=Rv1002c PE=3 SV=2 MVPVVSPGPLVPVADFGPLDRLRGWIVTGLITLLATVTRFLNLGSLTDAGTPIFDEKHYA PQAWQVLNNHGVEDNPGYGLVVHPPVGKQLIAIGEAIFGYNGFGWRFTGALLGVVLVALV VRIVRRISRSTLVGAIAGVLLICDGVSFVTARTALLDGFLTFFVVAAFGALIVDRDQVRE RMHIALLAGRSAATVWGPRVGVRWWRFGAGVLLGLACATKWSGVYFVLFFGAMALAFDVA ARRQYQVQRPWLGTVRRDVLPSGYALGLIPFAVYLATYAPWFASETAIDRHAVGQAVGRN SVVPLPDAVRSLWHYTAKAFHFHAGLTNSAGNYHPWESKPWTWPMSLRPVLYAIDQQDVA GCGAQSCVKAEMLVGTPAMWWLAVPVLAYAGWRMFVRRDWRYAVVLVGYCAGWLPWFADI DRQMYFFYAATMAPFLVMGISLVLGDILYHPGQGSERRTLGLIVVCCYVALVVTNFAWLY PVLTGLPISQQTWNLEIWLPSWR OST EC Number (BRENDA) 2.4.1.109 OST Genome Context O05586 Characterized Accessory Gene(s) Polyprenol-phosphate-mannose (PPM) synthase, Ppm1, is present. A second type of Ppm synthase (Rv3779 gene product) exclusive to slow-growing mycobacteria, is a membrane glycosyltransferase. It mannosylates polyprenyl-phosphates directly from GDP-mannose. PGL Additional Links CAZy Literatures Reference(s) 1) Scherman, H., Kaur, D., Pham, H., Skovierova, H., Jackson, M. and Brennan, P.J. (2009) Identification of a polyprenylphosphomannosyl synthase involved in the synthesis of mycobacterial mannosides. J Bacteriol, 191, 6769-6772. [PubMed: 19717608] 2) Lara, M., Servin-Gonzalez, L., Singh, M., Moreno, C., Cohen, I., Nimtz, M. and Espitia, C. (2004) Expression, secretion, and glycosylation of the 45- and 47-kDa glycoprotein of Mycobacterium tuberculosis in Streptomyces lividans. Appl Environ M Additional Comments Sec-mediated translocation influences the O-mannosylation. Ppm1 does not discriminate between polyprenol substrates with variable chain lengths and saturation of the isoprene units. Glycosylation sites have been found to be located within proline-rich domains (or Thr-rich sequences) near the N-terminus and the C-terminus. In a cell-free assay, the M. smegmatis mannosyltransferase activity in membrane and cell wall fraction has been shown to catalyze transfer of radiolabeled mannose fromGDP-[14C]mannose to peptide acceptors. The acceptors consisted of Thr-rich sequences from M. tuberculosis 45 kDa antigen (Ref. no. 3). The 45/47 kDa antigen (Apa) has also been glycosylated in Streptomyces lividans. The 45- and 47-kDa represent two glycoforms of the antigen (Ref. no. 2). Year of Identification 1989 Year of Validation 1996