Chemical and Biological Strategies for Engineering Cell Surface Glycosylation

Saxon, Eliana; Bertozzi, Carolyn R.

doi:10.1146/annurev.cellbio.17.1.1

Cited by 102 publications

(80 citation statements)

References 90 publications

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“…In our approach, the enzymatic reaction generates fluorescent products that become associated with the cell surface, resulting in a fluorescence profile representative of the catalytic selectivity of the displayed enzyme. Although we have capitalized on electrostatic interactions for product capture, a number of other methods for cell-surface modification (34,35) may be exploited for the capture of reaction products. In addition to the directed evolution of protease selectivity reported here, we believe that our methodology can be extended to other enzymes including various hydrolases and ligases.…”

Section: Discussionmentioning

confidence: 99%

Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity

Varadarajan

Gam

Olsen

et al. 2005

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

138

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The exquisite selectivity and catalytic activity of enzymes have been shaped by the effects of positive and negative selection pressure during the course of evolution. In contrast, enzyme variants engineered by using in vitro screening techniques to accept novel substrates typically display a higher degree of catalytic promiscuity and lower total turnover in comparison with their natural counterparts. Using bacterial display and multiparameter flow cytometry, we have developed a novel methodology for emulating positive and negative selective pressure in vitro for the isolation of enzyme variants with reactivity for desired novel substrates, while simultaneously excluding those with reactivity toward undesired substrates. Screening of a large library of random mutants of the Escherichia coli endopeptidase OmpT led to the isolation of an enzyme variant, 1.3.19, that cleaved an Ala-Arg peptide bond instead of the Arg-Arg bond preferred by the WT enzyme. Variant 1.3.19 exhibited greater than three million-fold selectivity (-Ala-Arg-͞-Arg-Arg-) and a catalytic efficiency for AlaArg cleavage that is the same as that displayed by the parent for the preferred substrate, Arg-Arg. A single amino acid Ser223Arg substitution was shown to recapitulate completely the unique catalytic properties of the 1.3.19 variant. These results can be explained by proposing that this mutation acts to ''swap'' the P 1 Arg side chain normally found in WT substrate peptides with the 223Arg side chain in the S 1 subsite of OmpT.engineering ͉ flow cytometry T he reprogramming of enzyme catalytic activity and selectivity is a central issue in protein biochemistry and biotechnology. Numerous structure-guided and directed evolution strategies have been used in search of enzyme variants that exhibit high catalytic rates with poor or inactive substrates of the parental enzyme (1-19). As impressive as these successes have been, the engineering of enzymes that exhibit turnover rates and selectivities with new substrates comparable to their natural counterparts has proven quite a challenge, especially when considering those enzymes for which a genetic selection strategy is not possible.In particular, enzymes engineered through laboratory evolution involving in vitro catalytic assays have often been found lacking, either with respect to turnover rates or selectivity, relative to catalyst-substrate pairs isolated from natural sources. As a typical example, an extensive directed evolution program led to the isolation of Escherichia coli ␤-glucuronidase variants with significant ␤-galactosidase (10) or xylanosidase (11) activities, but nonetheless even the best clones exhibited k cat ͞K m values Ͼ1,000 times lower than those of naturally occurring enzymes such as the E. coli ␤-galactosidase or the Thermoanaerobacterium saccharolyticum ␤-xylosidase.This trend appears to be general. In a recent comprehensive study, Aaron et al. (12) demonstrated that the evolution of higher activity toward poor substrates did not impair the parental catalytic activity, and,...

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Section: Discussionmentioning

confidence: 99%

Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity

Varadarajan

Gam

Olsen

et al. 2005

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

138

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“…A method for the selective labeling of sialoglycoproteins was developed by Saxon and Bertozzi (10); termed metabolic oligosaccharide engineering, it enables glycan labeling with probes for visualization in cells and enrichment of specific glycoconjugate types such as sialic acid for proteomic analysis (11)(12)(13). This technology involves metabolic labeling of glycans with a specifically reactive functional azido group, and the azido glycoproteins are then covalently tagged using an azide-specific chemical method, Staudinger ligation (14 -16).…”

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confidence: 99%

Targeted Identification of Sialoglycoproteins in Hypoxic Endothelial Cells and Validation in Zebrafish Reveal Roles for Proteins in Angiogenesis

Delcourt

Quevedo

Nonne

et al. 2015

Journal of Biological Chemistry

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Background: Target identification on tumor microvascularization is an opportunity for cancer therapy. Results: Membrane and secreted glycoproteins were identified on endothelial cells under hypoxia, and their function was assessed in zebrafish. Conclusion: Three novel hypoxia-regulated glycoproteins involved in angiogenesis have been identified and validated. Significance: We describe an approach that can be used to rapidly identify novel angiogenesis-related genes and their protein products.

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“…It is well known that glycans play important roles in cell-cell communication, intracellular signal transduction, protein folding, and stability (2,3).…”

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confidence: 99%

“…The major function of integrins is to connect cells to the extracellular matrix, activate intracellular signaling pathways, and regulate cytoskeletal formation (4). Integrin ␣5␤1 is well known as a fibronectin (FN) 3 receptor. The interaction between integrin ␣5 and FN is essential for cell migration, cell survival, and development (5)(6)(7)(8).…”

mentioning

confidence: 99%

An N-Glycosylation Site on theβ-Propeller Domain of the Integrin α5 Subunit Plays Key Roles in Both Its Function and Site-specific Modification byβ1,4-N-Acetylglucosaminyltransferase III

Sato

Isaji

Tajiri

et al. 2009

Journal of Biological Chemistry

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Recently we reported that N-glycans on the ␤-propeller domain of the integrin ␣5 subunit (S-3,4,5) are essential for ␣5␤1 heterodimerization, expression, and cell adhesion. Herein to further investigate which N-glycosylation site is the most important for the biological function and regulation, we characterized the S-3,4,5 mutants in detail. We found that site-4 is a key site that can be specifically modified by N-acetylglucosaminyltransferase III (GnT-III). The introduction of bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell adhesion and migration on fibronectin, whereas overexpression of N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration. The phenomenon is similar to previous observations that the functions of the wild-type ␣5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the ␣5 subunit could be duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in cell adhesion was consistently observed in the S-4,5 mutant but not in the S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a substantial decrease in erythroagglutinating phytohemagglutinin lectin staining and suppression of cell spread induced by GnT-III compared with that of either the site-3 single mutant or wild-type ␣5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the ␣5 subunit is the most important site for its biological functions. To our knowledge, this is the first demonstration that site-specific modification of N-glycans by a glycosyltransferase results in functional regulation.Glycosylation is a crucial post-translational modification of most secreted and cell surface proteins (1). Glycosylation is involved in a variety of physiological and pathological events, including cell growth, migration, differentiation, and tumor invasion. It is well known that glycans play important roles in cell-cell communication, intracellular signal transduction, protein folding, and stability (2, 3).Integrins comprise a family of receptors that are important for cell adhesion. The major function of integrins is to connect cells to the extracellular matrix, activate intracellular signaling pathways, and regulate cytoskeletal formation (4). Integrin ␣5␤1 is well known as a fibronectin (FN) 3 receptor. The interaction between integrin ␣5 and FN is essential for cell migration, cell survival, and development (5-8). In addition, integrins are N-glycan carrier proteins. For example, ␣5␤1 integrin contains 14 and 12 putative N-glycosylation sites on the ␣5 and ␤1 subunits, respectively. Several studies suggest that N-glycosylation is essential for functional integrin ␣5␤1. When human fibroblasts were cultured in the presence of 1-deoxymannojirimycin, which prevents N-linked oligosaccharide processing, immature ␣5␤1 integrin appeared on the cell surface, an...

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Chemical and Biological Strategies for Engineering Cell Surface Glycosylation

Cited by 102 publications

References 90 publications

Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity

Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity

Targeted Identification of Sialoglycoproteins in Hypoxic Endothelial Cells and Validation in Zebrafish Reveal Roles for Proteins in Angiogenesis

An N-Glycosylation Site on theβ-Propeller Domain of the Integrin α5 Subunit Plays Key Roles in Both Its Function and Site-specific Modification byβ1,4-N-Acetylglucosaminyltransferase III

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