e-journal

Rapid Assays for Lectin Toxicity and Binding Changes that Reflect Altered Glycosylation in Mammalian Cells

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Glycosylation engineering is used to generate glycoproteins, glycolipids, or proteoglycans
with a more defined complement of glycans on their glycoconjugates. For example,
a mammalian cell glycosylation mutant lacking a specific glycosyltransferase generates
glycoproteins, and/or glycolipids, and/or proteoglycans with truncated glycans missing
the sugar transferred by that glycosyltransferase, as well as those sugars that would be
added subsequently. In some cases, an alternative glycosyltransferase may then use the
truncated glycans as acceptors, thereby generating a new or different glycan subset in the
mutant cell. Another type of glycosylation mutant arises from gain-of-function mutations
that, for example, activate a silent glycosyltransferase gene. In this case, glycoconjugates
will have glycans with additional sugar(s) that are more elaborate than the glycans of wild
type cells. Mutations in other genes that affect glycosylation, such as nucleotide sugar
synthases or transporters, will alter the glycan complement in more general ways that
usually affect several types of glycoconjugates. There are now many strategies for generating
a precise mutation in a glycosylation gene in a mammalian cell. Large-volume
cultures of mammalian cells may also generate spontaneous mutants in glycosylation
pathways. This article will focus on how to rapidly characterize mammalian cells with
an altered glycosylation activity. The key reagents for the protocols described are plant
lectins that bind mammalian glycans with varying avidities, depending on the specific
structure of those glycans. Cells with altered glycosylation generally become resistant
or hypersensitive to lectin toxicity, and have reduced or increased lectin or antibody
binding. Here we describe rapid assays to compare the cytotoxicity of lectins in a lectin
resistance test, and the binding of lectins or antibodies by flow cytometry in a glycanbinding
assay. Based on these tests, glycosylation changes expressed by a cell can be
revealed, and glycosylation mutants classified into phenotypic groups that may reflect
a loss-of-function or gain-of-function mutation in a specific gene involved in glycan
synthesis.

Keywords: glycosylation mutants  mammalian cells  engineer glycans  glycan binding  lectins  antibodies  CHO cells

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Ketersediaan

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Informasi Detail

Judul Seri

Current Protocols in Chemical Biology

No. Panggil

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Penerbit

London : John Wiley & Sons, Inc.., 2014

Deskripsi Fisik

Curr. Protoc. Chem. Biol. 6:117-133

Bahasa

English

ISBN/ISSN

DOI: 10.1002/9780470

Klasifikasi

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Tipe Isi

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Tipe Media

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Tipe Pembawa

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Edisi

6:June 2014

Subjek

KIMIA

Info Detail Spesifik

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Pernyataan Tanggungjawab

agus

Versi lain/terkait

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