Peptide-sugar ligation catalyzed by transpeptidase sortase: A facile approach to neoglycoconjugate synthesis

Article abstract of DOI:10.1021/ja077358g

Glycoconjugate synthesis involving sugar and polypeptide remains a formidable challenge. Here we report a novel enzymatic method involving an unprecedented sortase-catalyzed transamidation reaction for site-specific engineering of sugars into native prote

Full text of DOI:10.1021/ja077358g

Published on Web 01/30/2008
Peptide-Sugar Ligation Catalyzed by Transpeptidase Sortase: A Facile
Approach to Neoglycoconjugate Synthesis
Sharmishtha Samantaray, Uttara Marathe, Sayani Dasgupta, Vinay K. Nandicoori, and
Rajendra P. Roy*
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India
Received September 23, 2007; E-mail: rproy@nii.res.in
Development of new methods for linking sugars to peptides or
proteins is an active area of research because natural glycopeptides
or neoglycoconjugates play important roles in biology and medicine
and are indispensable tools for probing several biological processes.1-4
However, despite dramatic progress in synthetic carbohydrate and
protein chemistry in recent years, glycoconjugate synthesis involv-
ing sugar and polypeptide remains a formidable task. This is
principally because synthetic protocols are quite demanding and
involve multiple reaction steps with requirements of rather extensive
protection of reactive functionalities. The problem may be in part
or completely obviated through the intermediary of enzymes.
Indeed, glycosidases and glycosyl transferases, in appropriate
situations, have made the synthesis of oligosaccharides much
simpler.5-7 Given the current ease with which peptides are
assembled by solid phase methodology and proteins obtained from
expression systems, the availability of enzymes capable of co-
valently linking a presynthesized sugar and a polypeptide would
greatly facilitate the convergent semisynthesis of glycoconjugates
with exquisite biological properties. We report here a novel
enzymatic approach, using an unprecedented sortase-catalyzed
transamidation reaction, for the facile one-pot synthesis of glyco-
conjugates comprising amino sugars and native polypeptides.
The transpeptidase sortase, present in the cell envelope of most
gram-positive bacteria, catalyzes the covalent anchoring of several
bacterial surface proteins to the peptidoglycan cross-bridges of the
cell wall.8,9 Sortase A of Staphylococcus aureus recognizes a
LPXTG pentapeptide sequence motif located near the C-terminus
of the target proteins, cleaves at Thr-Gly peptide bond, and
catalyzes the formation of a new peptide bond between the threonyl
carboxyl and amino group of the peptidoglycan pentaglycine cross-
bridges.10 The transpeptidation reaction proceeds in two steps
without the aid of any extraneous molecule; the active site cysteine
residue first attacks the target LPXTG substrate forming an acyl-
enzyme intermediate which in the second step is resolved by the
nucleophilic attack of the amino group of the terminal Gly residue
of the peptidoglycan. In the absence of a suitable amino nucleophile,
the LPXTG peptide substrate is slowly hydrolyzed. Sortase-
mediated transpeptidation reaction involving LPXTG and ami-
noglycine containing polypeptides proceeds smoothly in vitro and
has been applied to the synthesis of protein-peptide and peptide-
nucleic acid conjugates that would have been difficult to obtain by
purely chemical or genetic means.11-13 Interestingly, ligation of
LPXTG substrates can occur even with polypeptides containing a
single Gly residue at the amino terminus. This observation of
relaxed specificity for the amine nucleophile and the fact that the
transpeptidation reaction does not require intermediary of high-
energy phosphates prompted us to explore sortase-mediated ligation
of polypeptides to amino sugars with a view to develop an
enzymatic approach to glycoconjugate synthesis.
We considered 6-aminohexoses as potential sugar substrates with
the idea that the -CH2-NH2 moiety present in these sugars might
mimic some elements of the glycine structure. Accordingly, we
tested the potential of sortase to ligate 6-deoxy-6-aminoglucose and
6-deoxy-6-aminomannose to a model YALPETGK peptide sub-
strate. HPLC assays followed by MALDI-TOF or ESMS analyses
revealed the formation of respective YALPET-sugar adducts,
suggesting that the above amino sugars indeed acted as nucleophiles
in the transamidation reaction (Supporting Information Figure 1).
In contrast, the substrate peptide was hydrolyzed to YALPET
without the formation of the YALPET-sugar adduct when glu-
cosamine was used as a substrate. Consistent with the known
tolerance of sortase for the LPXTG recognition motif,14 the
YALPMTGK peptide sequence also reacted with 6-deoxy-6-
aminoglucose or 6-deoxy-6-aminomannose but not with glu-
cosamine.
To further probe the specificity requirements as well as to see if
6-aminohexoses can serve as recognition tags for peptide-sugar
ligations, we investigated the ability of sortase to ligate peptides
to an aminoglycoside class of therapeutically important antibiotics.
These antibiotics are built up by a variety of amino sugars of the
6-amino or the 2,6-diamino type besides containing several other
amino functionalities15 (Figure 1). The central scaffold of ami-
noglycoside antibiotics is the 2-deoxystreptamine ring to which
amino sugars are substituted at positions 4 and 6 (as in tobramycin
and kanamycins) or 4 and 5 (as in ribostamycin, neomycin, and
paromomycin). Sortase-mediated ligation of model peptide sub-
strates to aminoglycoside antibiotics proceeded smoothly. Analyses
of the reaction products by reversed phase HPLC (Figure 2)
followed by MALDI (Supporting Information Table 2) revealed
the formation of specific conjugates between antibiotics and peptides
in the yields varying from 35 to 70% for the kanamycin class, and
about 18-30% for the ribostamycin class of antibiotics. Electro-
spray mass spectrometry (Supporting Information Figures 2-5) of
the respective conjugates produced fragmentations that unambigu-
ously showed occurrence of peptide ligation exclusively at a single
6-amino site in ring A of kanamycins, tobramycin, and ribostamycin
or ring D of paromomycin and neomycin. Thus, conjugation of
peptide substrates was limited to the 6-amino site in the antibiotics
despite the presence of a plethora of amino groups, indicating rather
strict specificity and selectivity for the sugar amino groups by
sortase.
Next, we explored conjugation of biologically relevant peptides
to aminoglycoside antibiotics. Toward this, we considered peptide
sequences derived from or based on Tat and Rev proteins of HIV
because these proteins play important roles in virus replication
through their interactions with structured viral RNA target sites,
TAR in the case of Tat and RRE in the case of Rev.16 Interestingly,
aminoglycoside antibiotics and analogues,17 as well as short
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10.1021/ja077358g CCC: $40.75 © 2008 American Chemical Society

C O M M U N I C A T I O N S
(Mrp protein, NP_372281) from Staphylococcus aureus nested with
a LPNTG sequence motif in its carboxy terminal region. We
employed HPLC assays and MALDI-TOF to investigate the ligation
of tobramycin to Mrp (Supporting Information Figures 8 and 9).
Incubation of Mrp alone with sortase led to generation of two
fragments expected from hydrolysis at the T-G peptide bond.
However, incubation of the protein with sortase in the presence of
tobramycin produced specific conjugate (Mrp-LPNT-tobramycin)
with a yield of more than 45% in 6 h.
In summary, our work demonstrates that sortase can transfer
peptide substrates to oligosaccharides appended with a 6-deoxy-
6-aminohexose moiety in a selective manner as that of an
oligoglycine sequence. Such an enzymatic activity of peptide-sugar
ligation, presumably promiscuous in origin for sortase, is hitherto
unknown. This robust reaction provides a simple and straightforward
method for covalent ligation of a prefabricated sugar containing a
6-aminohexose tag to synthetic peptides and expressed proteins
encoded with a C-terminal LPXTG sortase recognition sequence.
We envision several biotechnological applications of this methodol-
ogy, including generation of novel glycopeptide-based immuno-
vaccines comprising oligosaccharides substituted with multiple
peptides and glycolabeling of proteins. Besides, the facile assembly
of aminoglycoside antibiotic conjugates offers tremendous pos-
sibilities of generating new RNA ligands and therapeutics. The work
on anti-HIV activity of antibiotics-Tat/Rev conjugates is in
progress.
Figure 1. (a) General peptide ligation reaction catalyzed by sortase. (b)
LPXTG peptide substrates used in the study. (c) Amino sugars used in the
study. The 6-amino site is shown in blue.
Acknowledgment. We thank Gokhale Laboratory and Dr. P.
Sahai for help with mass measurements. This work was supported
by NII core, National Bioscience Award, and DBT-ICMR grants
to R.P.R. from the Department of Biotechnology, India.
Supporting Information Available: Peptide-sugar ligations,
product characterization, and RNA binding data. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Figure 2. Sortase-catalyzed conjugation of YALPETGK model peptide
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Finally, we tested the feasibility of using sortase for site-specific
conjugation of sugars to proteins. For this, we expressed a protein
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