A novel enzymatic approach was developed for facile production of glycopeptides carrying natural eukaryotic N-glycans. In this approach, peptides can be GlcNAcylated at one or two natural N-glycosylation sites via two-step enzymatic reactions catalyzed by an evolved N-glycosyltransferase (ApNGT) and a glucosamine N-acetyltransferase (GlmA), respectively. The resulting GlcNAc-peptides were further modified by an endo-β-N-acetylglucosaminidase M mutant (EndoM) to generate glycopeptides. In three steps of enzymatic catalysis, glycopeptides carrying complex-type N-glycans can be efficiently synthesized.
Naturally occurring -glycoproteins exhibit glycoform heterogeneity with respect to-glycan sequon occupancy (macroheterogeneity) and glycan structure (microheterogeneity). However, access to well-defined glycoproteins is always important for both basic research and therapeutic purposes. As a result, there has been a substantial effort to identify and understand the catalytic properties of -glycosyltransferases, enzymes that install the first glycan on the protein chain. In this study we found that ApNGT, a newly discovered cytoplasmic-glycosyltransferase from , has strict selectivity toward the residues around the Asn of-glycosylation sequon by screening a small library of synthetic peptides. The inherent stringency was subsequently demonstrated to be closely associated with a critical residue (Gln-469) of ApNGT which we propose hinders the access of bulky residues surrounding the occupied Asn into the active site. Site-saturated mutagenesis revealed that the introduction of small hydrophobic residues at the site cannot only weaken the stringency of ApNGT but can also contribute to enormous improvement of glycosylation efficiency against both short peptides and proteins. We then employed the most efficient mutant (Q469A) other than the wild-type ApNGT to produce a homogeneous glycoprotein carrying multiple (up to 10) -glycans, demonstrating that this construct is a promising biocatalyst for potentially addressing the issue of macroheterogeneity in glycoprotein preparation.
Neu5Ac,
Neu5Gc, and KDN are three forms of sialic acids in vertebrates
that possess distinct biological functions. Herein, we report the
synthesis and metabolic incorporation of the 9-azido analogues of
three sialic acid forms in mammalian cells. The incorporated sialic
acid analogues enable fluorescent imaging of cell-surface sialoglycans
and proteomic profiling of sialoglycoproteins. Furthermore, we apply
them to metabolically engineer cell surfaces with sialoglycans terminated
with distinct sialic acids or their 9-azido analogues. The remodeled
cells expressing specific cell-surface sialoglycoforms show distinct
binding affinity toward subtilase cytotoxin (SubAB), a toxin secreted
by Shiga toxigenic Escherichia coli. The 9-azido
analogues of sialic acid forms developed in this work provide a versatile
tool for metabolic remodeling of cell-surface properties and modulating
pathogen–host interactions.
In
this study, an accurately and digitally regulated allosteric
nanoswitch based on the conformational control of two DNA hairpins
was developed. By switching between UV irradiation and blue light
conditions, the second molecular beacon (H#2) would bind/separate
with a repression sequence (RES) via the introduced PTG molecules
(a photosensitive azobenzene derivative), resulting in the target
aptamer sequence in the first molecular beacon (H#1) not being able/being
able to hold the stem-loop configuration, hence losing/regaining the
ability to bind with the target. Importantly, we successfully monitor
conformation changes of the nanoswitch by an elegant mathematical
model for connecting K
i (the dissociation
constant between RES and H#2) with K
d (the
overall equilibrium constant of the nanoswitch binding the target),
hence realizing “observing” DNA structure across dimensions
from “structural visualization” to digitization and,
accurately, digitally regulating DNA structure from digitization to
“structural visualization”.
Despite the absence of any homologs of Tannerella forsythia KLIKK proteases in Tannerella sp.6_1_58FAA_CT1, the strain possesses a putative cysteine protease (G9S4N1) closely related to RgpB of Porphyromonas gingivalis. G9S4N1 lacks obvious propeptide that behaves as inhibitor of proteases and was proven to be a propeptide-independent protease. Unlike RgpB, which exclusively cleaves ArgXaa bonds, G9S4N1 exhibits both arginine- and citrulline-specific activities. Mutations of Asp177, a potential P1-Arg binding site, to uncharged or positively charged residues did not alter the substrate specificity of G9S4N1 significantly. Moreover, a group of arginine-specific proteases from different species including porcine trypsin, bovine thrombin, and a trypsin-like serine protease of dengue 2 virus CF40-Gly-NS3pro185 also display different specificity toward citrulline residue, suggesting that citrulline-modified protein might have different roles and destiny in biological processes involving various proteases.
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