Peptide−Sugar Ligation Catalyzed by Transpeptidase Sortase:  A Facile Approach to Neoglycoconjugate Synthesis

Samantaray, Sharmishtha; Marathe, Uttara; Dasgupta, Sayani; Nandicoori, Vinay Kumar; Roy, Rajendra P.

doi:10.1021/ja077358g

Cited by 103 publications

(107 citation statements)

References 19 publications

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“…We used sortase to covalently attach protein antigens (i.e., sfGFP, mCherry, and fHbp) to the surface of preassembled, purified VipA/B sheaths in vitro under native conditions. Other groups have exploited sortase in vitro to label proteins with chemical moieties, such as fluorescent probes (27,28), lipids (29), sugars (30), and proteins (31). Sortase may be applied to decorate sheaths with appropriate chemical ligands or adhesins that might give sheaths binding activity for particular target cells, Total mouse IgG ELISA titers are reported as absorbency units at OD 405 for the same serum dilution 1:32,000.…”

Section: Discussionmentioning

confidence: 99%

Type VI secretion system sheaths as nanoparticles for antigen display

Tordello

Danilchanka

McCluskey

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The bacterial type 6 secretion system (T6SS) is a dynamic apparatus that translocates proteins between cells by a mechanism analogous to phage tail contraction. T6SS sheaths are cytoplasmic tubular structures composed of stable VipA-VipB (named for ClpV-interacting protein A and B) heterodimers. Here, the structure of the VipA/B sheath was exploited to generate immunogenic multivalent particles for vaccine delivery. Sheaths composed of VipB and VipA fused to an antigen of interest were purified from Vibrio cholerae or Escherichia coli and used for immunization. Sheaths displaying heterologous antigens generated better immune responses against the antigen and different IgG subclasses compared with soluble antigen alone. Moreover, antigen-specific antibodies raised against sheaths presenting Neisseria meningitidis factor H binding protein (fHbp) antigen were functional in a serum bactericidal assay. Our results demonstrate that multivalent nanoparticles based on the T6SS sheath represent a versatile scaffold for vaccine applications.vaccine delivery | type 6 secretion system | nanoparticles | antigen | immunogen

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Section: Discussionmentioning

confidence: 99%

Type VI secretion system sheaths as nanoparticles for antigen display

Tordello

Danilchanka

McCluskey

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…18 In addition, recent protein/peptide-protein/peptide oligomerization 12 and labeling of proteins on cells 19 have demonstrated its applicability toward biological use. The present work is the first report of sortase-mediated ligation of antibodies, or indeed coupling of any targeting protein, and the first demonstration of the in vivo use of the sortase technology.…”

Section: Discussionmentioning

confidence: 99%

Enzymatic Single-Chain Antibody Tagging

Prabhu

Leitner

et al. 2011

Circulation Research

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Objective: To develop a biochemically robust, highly reproducible, and site-specific coupling method using the Staphylococcus aureus sortase A enzyme for the conjugation of a single-chain antibody (scFv) to nanoparticles and cells for molecular imaging and cell homing in cardiovascular diseases. This scFv specifically binds to activated platelets, which play a pivotal role in thrombosis, atherosclerosis, and inflammation. Methods and Results:

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“…2 sequence corresponding to amino acids 60 to 204 was expressed and purified as described previously (34). Briefly, Escherichia coli BL21(DE3) cells containing the srtA plasmid were grown in LB broth at 37°C until the optical density at 600 nm reached 0.6 and then the cells were induced with 0.2 mM isopropyl 1-thio-␤-D-galactopyranoside at 30°C for 3 h. The cells were then harvested, suspended in buffer A (10 mM Tris⅐HCl (pH 7.5), 40 mM NaCl, 1 mM 2-mercaptoethanol), and lysed by sonication.…”

Section: Srtamentioning

confidence: 99%

“…Given the limited synthetic utility of pilin sortases, we turned our attention toward SrtA of S. aureus and explored if this enzyme could accept ⑀-amino group of Lys residue in the transpeptidation reaction. We were motivated by the fact that SrtA displays rather relaxed specificity for the amine nucleophile and can accept a variety of molecules, terminating in aminomethyl (H 2 NCH 2 -) or aminoethyl (H 2 NCH 2 CH 2 -) moieties, as substitutes for oligoglycines (28,31,34).…”

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confidence: 99%

Isopeptide Ligation Catalyzed by Quintessential Sortase A

Dasgupta

Samantaray

Sahal

et al. 2011

Journal of Biological Chemistry

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The housekeeping transpeptidase sortase A (SrtA) from Staphyloccocus aureus catalyzes the covalent anchoring of surface proteins to the cell wall by linking the threonyl carboxylate of the LPXTG recognition motif to the amino group of the pentaglycine cross-bridge of the peptidoglycan. SrtA-catalyzed ligation of an LPXTG containing polypeptide with an aminoglycine-terminated moiety occurs efficiently in vitro and has inspired the use of this enzyme as a synthetic tool in biological chemistry. Here we demonstrate the propensity of SrtA to catalyze "isopeptide" ligation. Using model peptide sequences, we show that SrtA can transfer LPXTG peptide substrates to the ⑀-amine of specific Lys residues and form cyclized and/or a gamut of branched oligomers. Our results provide insights about principles governing isopeptide ligation reactions catalyzed by SrtA and suggest that although cyclization is guided by distance relationship between Lys (⑀-amine) and Thr (␣-carboxyl) residues, facile branched oligomerization requires the presence of a stable and long-lived acyl-enzyme intermediate.

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Peptide−Sugar Ligation Catalyzed by Transpeptidase Sortase: A Facile Approach to Neoglycoconjugate Synthesis

Cited by 103 publications

References 19 publications

Type VI secretion system sheaths as nanoparticles for antigen display

Type VI secretion system sheaths as nanoparticles for antigen display

Enzymatic Single-Chain Antibody Tagging

Isopeptide Ligation Catalyzed by Quintessential Sortase A

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