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The highly conserved domain of unknown function 1792 has a distinct glycosyltransferase fold.

Zhang H, Zhu F, Yang T, Ding L, Zhou M, Li J, Haslam SM, Dell A, Erlandsen H, Wu H - Nat Commun (2014)

Bottom Line: Structural studies, however, have only revealed two distinct glycosyltransferase (GT) folds, GT-A and GT-B.Biochemical studies reveal that the domain is a glucosyltransferase, and it catalyses the transfer of glucose to the branch point of the hexasaccharide O-linked to the serine-rich repeat of the bacterial adhesin, Fap1 of Streptococcus parasanguinis.Thus, DUF1792 represents a new family of glycosyltransferases; therefore, we designate it as a GT-D glycosyltransferase fold.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatric Dentistry, Microbiology, Schools of Dentistry and Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

ABSTRACT
More than 33,000 glycosyltransferases have been identified. Structural studies, however, have only revealed two distinct glycosyltransferase (GT) folds, GT-A and GT-B. Here we report a 1.34-Å resolution X-ray crystallographic structure of a previously uncharacterized 'domain of unknown function' 1792 (DUF1792) and show that the domain adopts a new fold and is required for glycosylation of a family of serine-rich repeat streptococcal adhesins. Biochemical studies reveal that the domain is a glucosyltransferase, and it catalyses the transfer of glucose to the branch point of the hexasaccharide O-linked to the serine-rich repeat of the bacterial adhesin, Fap1 of Streptococcus parasanguinis. DUF1792 homologues from both Gram-positive and Gram-negative bacteria also exhibit the activity. Thus, DUF1792 represents a new family of glycosyltransferases; therefore, we designate it as a GT-D glycosyltransferase fold. As the domain is highly conserved in bacteria and not found in eukaryotes, it can be explored as a new antibacterial target.

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Mass spectrometric analysis of rFap1 glycanThe glycans were reductively eliminated from the protein, and permethylated.Derivatized glycans were purified on reverse phase C18 Sep-Pak® columns. The MALDI-TOF spectra of the 35% acetonitrile (MeCN) eluate (a), the 50% MeCN eluate (b), and MALDI-TOF/TOF spectrum of the peak at mass-to-charge ratio (m/z) 1361.6 (c) are shown. In spectra (a) and (b), peaks corresponding to sodiated glycans are colored red and annotated with m/z and glycan structures. Other black signals are due to under-permethylation (minus 14 in m/z) or contaminations from the matrix. In spectra (c), peaks corresponding to diagnostic fragments are annotated with m/z and glycan structure. Notably, the peak at m/z 506.4 corresponds to a double-cleaved fragment, indicating the fully synthesized glycan is branched.
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Figure 1: Mass spectrometric analysis of rFap1 glycanThe glycans were reductively eliminated from the protein, and permethylated.Derivatized glycans were purified on reverse phase C18 Sep-Pak® columns. The MALDI-TOF spectra of the 35% acetonitrile (MeCN) eluate (a), the 50% MeCN eluate (b), and MALDI-TOF/TOF spectrum of the peak at mass-to-charge ratio (m/z) 1361.6 (c) are shown. In spectra (a) and (b), peaks corresponding to sodiated glycans are colored red and annotated with m/z and glycan structures. Other black signals are due to under-permethylation (minus 14 in m/z) or contaminations from the matrix. In spectra (c), peaks corresponding to diagnostic fragments are annotated with m/z and glycan structure. Notably, the peak at m/z 506.4 corresponds to a double-cleaved fragment, indicating the fully synthesized glycan is branched.

Mentions: We employed a variety of mass spectrometric glycomic strategies to characterize Fap1 glycosylation. Because it was difficult to isolate native Fap1 in sufficient quantities for in-depth structure analysis, we first characterized the glycosylation of recombinant Fap1 which we obtained by co-expression of recombinant Fap1 (rFap1)35 with all the glycosyltransferases identified from the fap1 locus. rFap1 was purified and subjected to beta-elimination to release the O-linked glycans for MS analysis. MALDI-TOF mass fingerprinting (Fig. 1a and b) of the beta-eliminated permethylated glycans showed a mixture of glycans ranging in size from a monosaccharide (hexose) up to a hexasaccharide comprised of one deoxyhexose, two HexNAcs and three hexoses. The latter is consistent with a previously reported monosaccharide composition for the native Fap1 glycan22. The smaller glycans correspond to biosynthetic precursors. Each peak from the glycan fingerprint was further analyzed by MALDI-TOF/TOF to generate glycan sequences. The MS/MS spectrum of the hexasaccharide peak at m/z 1361.6 is shown in Fig. 1(c). The data are fully consistent with the branched structure shown in the cartoon annotation on this figure. The identities of the sugars and their linkages were determined by additional GC-EI-MS experiments. Sugar linkage analysis of partially methylated alditol acetates (Supplementary Table 1) determined rhamnose and glucose as non-reducing sugars in the hexasaccharide, and identified the reducing sugar as 6-linked GlcNAc. Other linkages observed were 3-linked GlcNAc, and 3- and 2,6-linked Glc, the latter being consistent with the branched sequence shown in Fig. 1(c).


The highly conserved domain of unknown function 1792 has a distinct glycosyltransferase fold.

Zhang H, Zhu F, Yang T, Ding L, Zhou M, Li J, Haslam SM, Dell A, Erlandsen H, Wu H - Nat Commun (2014)

Mass spectrometric analysis of rFap1 glycanThe glycans were reductively eliminated from the protein, and permethylated.Derivatized glycans were purified on reverse phase C18 Sep-Pak® columns. The MALDI-TOF spectra of the 35% acetonitrile (MeCN) eluate (a), the 50% MeCN eluate (b), and MALDI-TOF/TOF spectrum of the peak at mass-to-charge ratio (m/z) 1361.6 (c) are shown. In spectra (a) and (b), peaks corresponding to sodiated glycans are colored red and annotated with m/z and glycan structures. Other black signals are due to under-permethylation (minus 14 in m/z) or contaminations from the matrix. In spectra (c), peaks corresponding to diagnostic fragments are annotated with m/z and glycan structure. Notably, the peak at m/z 506.4 corresponds to a double-cleaved fragment, indicating the fully synthesized glycan is branched.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4352575&req=5

Figure 1: Mass spectrometric analysis of rFap1 glycanThe glycans were reductively eliminated from the protein, and permethylated.Derivatized glycans were purified on reverse phase C18 Sep-Pak® columns. The MALDI-TOF spectra of the 35% acetonitrile (MeCN) eluate (a), the 50% MeCN eluate (b), and MALDI-TOF/TOF spectrum of the peak at mass-to-charge ratio (m/z) 1361.6 (c) are shown. In spectra (a) and (b), peaks corresponding to sodiated glycans are colored red and annotated with m/z and glycan structures. Other black signals are due to under-permethylation (minus 14 in m/z) or contaminations from the matrix. In spectra (c), peaks corresponding to diagnostic fragments are annotated with m/z and glycan structure. Notably, the peak at m/z 506.4 corresponds to a double-cleaved fragment, indicating the fully synthesized glycan is branched.
Mentions: We employed a variety of mass spectrometric glycomic strategies to characterize Fap1 glycosylation. Because it was difficult to isolate native Fap1 in sufficient quantities for in-depth structure analysis, we first characterized the glycosylation of recombinant Fap1 which we obtained by co-expression of recombinant Fap1 (rFap1)35 with all the glycosyltransferases identified from the fap1 locus. rFap1 was purified and subjected to beta-elimination to release the O-linked glycans for MS analysis. MALDI-TOF mass fingerprinting (Fig. 1a and b) of the beta-eliminated permethylated glycans showed a mixture of glycans ranging in size from a monosaccharide (hexose) up to a hexasaccharide comprised of one deoxyhexose, two HexNAcs and three hexoses. The latter is consistent with a previously reported monosaccharide composition for the native Fap1 glycan22. The smaller glycans correspond to biosynthetic precursors. Each peak from the glycan fingerprint was further analyzed by MALDI-TOF/TOF to generate glycan sequences. The MS/MS spectrum of the hexasaccharide peak at m/z 1361.6 is shown in Fig. 1(c). The data are fully consistent with the branched structure shown in the cartoon annotation on this figure. The identities of the sugars and their linkages were determined by additional GC-EI-MS experiments. Sugar linkage analysis of partially methylated alditol acetates (Supplementary Table 1) determined rhamnose and glucose as non-reducing sugars in the hexasaccharide, and identified the reducing sugar as 6-linked GlcNAc. Other linkages observed were 3-linked GlcNAc, and 3- and 2,6-linked Glc, the latter being consistent with the branched sequence shown in Fig. 1(c).

Bottom Line: Structural studies, however, have only revealed two distinct glycosyltransferase (GT) folds, GT-A and GT-B.Biochemical studies reveal that the domain is a glucosyltransferase, and it catalyses the transfer of glucose to the branch point of the hexasaccharide O-linked to the serine-rich repeat of the bacterial adhesin, Fap1 of Streptococcus parasanguinis.Thus, DUF1792 represents a new family of glycosyltransferases; therefore, we designate it as a GT-D glycosyltransferase fold.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatric Dentistry, Microbiology, Schools of Dentistry and Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

ABSTRACT
More than 33,000 glycosyltransferases have been identified. Structural studies, however, have only revealed two distinct glycosyltransferase (GT) folds, GT-A and GT-B. Here we report a 1.34-Å resolution X-ray crystallographic structure of a previously uncharacterized 'domain of unknown function' 1792 (DUF1792) and show that the domain adopts a new fold and is required for glycosylation of a family of serine-rich repeat streptococcal adhesins. Biochemical studies reveal that the domain is a glucosyltransferase, and it catalyses the transfer of glucose to the branch point of the hexasaccharide O-linked to the serine-rich repeat of the bacterial adhesin, Fap1 of Streptococcus parasanguinis. DUF1792 homologues from both Gram-positive and Gram-negative bacteria also exhibit the activity. Thus, DUF1792 represents a new family of glycosyltransferases; therefore, we designate it as a GT-D glycosyltransferase fold. As the domain is highly conserved in bacteria and not found in eukaryotes, it can be explored as a new antibacterial target.

Show MeSH