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Harnessing the acceptor substrate promiscuity of Clostridium botulinum Maf glycosyltransferase to glyco-engineer mini-flagellin protein chimeras.
Sunsunwal, Sonali; Khairnar, Aasawari; Subramanian, Srikrishna; Ramya, T N C.
Affiliation
  • Sunsunwal S; CSIR- Institute of Microbial Technology, Sector 39-A, Chandigarh, 160036, India.
  • Khairnar A; CSIR- Institute of Microbial Technology, Sector 39-A, Chandigarh, 160036, India.
  • Subramanian S; CSIR- Institute of Microbial Technology, Sector 39-A, Chandigarh, 160036, India.
  • Ramya TNC; CSIR- Institute of Microbial Technology, Sector 39-A, Chandigarh, 160036, India. ramya@imtech.res.in.
Commun Biol ; 7(1): 1029, 2024 Aug 21.
Article in En | MEDLINE | ID: mdl-39169227
ABSTRACT
Several bacterial flagellins are O-glycosylated with nonulosonic acids on surface-exposed Serine/Threonine residues by Maf glycosyltransferases. The Clostridium botulinum Maf glycosyltransferase (CbMaf) displays considerable donor substrate promiscuity, enabling flagellin O-glycosylation with N-acetyl neuraminic acid (Neu5Ac) and 3-deoxy-D-manno-octulosonic acid in the absence of the native nonulosonic acid, a legionaminic acid derivative. Here, we have explored the sequence/structure attributes of the acceptor substrate, flagellin, required by CbMaf glycosyltransferase for glycosylation with Neu5Ac and KDO, by co-expressing C. botulinum flagellin constructs with CbMaf glycosyltransferase in an E. coli strain producing cytidine-5'-monophosphate (CMP)-activated Neu5Ac, and employing intact mass spectrometry analysis and sialic acid-specific flagellin biotinylation as readouts. We found that CbMaf was able to glycosylate mini-flagellin constructs containing shortened alpha-helical secondary structural scaffolds and reduced surface-accessible loop regions, but not non-cognate flagellin. Our experiments indicated that CbMaf glycosyltransferase recognizes individual Ser/Thr residues in their local surface-accessible conformations, in turn, supported in place by the secondary structural scaffold. Further, CbMaf glycosyltransferase also robustly glycosylated chimeric proteins constructed by grafting cognate mini-flagellin sequences onto an unrelated beta-sandwich protein. Our recombinant engineering experiments highlight the potential of CbMaf glycosyltransferase in future glycoengineering applications, especially for the neo-O-sialylation of proteins, employing E. coli strains expressing CMP-Neu5Ac (and not CMP-KDO).
Subject(s)

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Glycosyltransferases / Clostridium botulinum / Flagellin Language: En Journal: Commun Biol Year: 2024 Document type: Article Affiliation country: India Country of publication: United kingdom

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Glycosyltransferases / Clostridium botulinum / Flagellin Language: En Journal: Commun Biol Year: 2024 Document type: Article Affiliation country: India Country of publication: United kingdom