Post-Translational Modifications

Higher eukaryotes perform a variety of post-translational modifications, including methylation, sulfation, phosphorylation, lipid addition, and glycosylation. Such modifications may be of critical importance to the function of an expressed protein. Secreted proteins, membrane proteins, and proteins targeted to vesicles or certain intracellular organelles are likely to be glycosylated. The most common and best studied is N-linked glycosylation, where oligosaccharides are uniquely added to asparagine found in Asn-X-Ser/Thr recognition sequences in proteins. Another type of glycosylation is O-linked glycosylation, which involves either simple oligosaccharide chains or glycosaminoglycan chains (1). When expressing and purifying a glycosylated protein in a heterologous expression system, it may be desirable to quickly determine whether the protein is glycosylated properly. Protocols for carbohydrate analysis of proteins have been published to allow the molecular biologist to characterize glycosylated proteins of interest (2). The following sections discuss glycosylation patterns found in eukaryotic cells. Glycosylation in Mammalian Cells N-linked glycoproteins contain standard branched structures, which are composed of mannose (Man), galactose, N-acetylglucosamine (GlcNAc) and neuramic acids. O-linked glycoproteins are composed of various number of sugars including galactose, N-acetylglucosamine, N-acetylgalactosamine, and neuramic acids. Glycosylation in Insect Cells The nature of N-linked glycosylation in insect cells (Sf21, Sf9, High Five) is dependent on the protein expressed and the host cell line. N-linked glycosylation is generally of the high-mannose type. O-linked glycosylation is similar, although not identical, to mammalian cells, depending on localization and type of protein. Drosophila N-linked glycosylation is less complex in that it is not trimmed and sialylated. Thus Drosophila proteins have a high mannose content. Drosophila can also add O-linked glycosylation. Mimic Sf9 Insect Cells are modified Sf9 cells that stably express a variety of mammalian glycosyltransferases. These enzymes allow for production of biantennary, terminally sialyated N-glycans from insect cells. The cells can be used to produce more mammalian-like proteins in both baculovirus and stable insect expression systems. See page XX or contact Technical Service for more information about this cell line. Glycosylation in Yeast S. cerevisiae N-linked glycoproteins contain only highly branched and extended high mannose structures (hyperglycosylation). S. cerevisiae O-linked glycoproteins are composed of less than four mannose residues. Pichia N-linked glycosylation consists mostly of short chain Man (3) GlcNAc residues and is closer to the typical mammalian high-mannose glycosylation pattern. Pichia O-linked oligosaccharides are present but are not major components of the total soluble glycoprotein of Pichia. Following you will find: an outline of the two basic types of N-linked glycosylation (1,2), a table of glycosylation inhibitors that can be used in vivo (2), and a table of enzymes which can be used to analyze carbohydrate structure on proteins. For further information about glycosylation in eukaryotes, see reference 4.

Man

Man

Glc NAC Man

Man
Swainsonine

O NH Glc NAC C CH2

C CH HN

Asn

Man
Deoxymannojirimycin Constanospermine

Glc NAC

Glc NAC Man Man

Tunicamycin Monosaccharides - removed in endoplasmic reticulum (ER) Glucosidase I and II ER mannosidase Monosaccharides - removed in Golgi Apparatus -mannosidase I and II Added in Golgi Apparatus Core oligosaccharides Tunicamycin prevents synthesis of this oligosaccharide

GLC

Man

GLC

GLC

Man
Deoxymannojirimycin

Complex N-linked Glycosylation

Glc NAC NH Man Glc NAC Glc NAC Man Man Glc NAC Gal NeuAc Glc NAC Gal NeuAc Gal NeuAc

High Mannose N-linked Glycosylation

Man NH Man Glc NAC Glc NAC Man Man Man

Man

Asn
X Ser/Thr C O

Asn
X Ser/Thr C O

Abbreviations: Asn: Gal: Ser: Thr: Man: Asparagine Galactose Serine Threonine Mannose

GlcNAc: NeuAc:

N-acetylglucosamine N-acetylneuraminic acid

Effects of Certain Antibiotics on Glycosylation Structure in Eukaryotes (2) Effects on Electrophoretic Mobility in SDS-PAGE Proteins migrate faster and show less heterogeneity

Type of Inhibitor Tunicamycin

Target Enzyme GlcNAc transferase

Effects on Oligosaccharide Structure Prevents first synthetic step of the core oligosaccharide Glycosylation of Asn residues does not occur Prevents removal of first glucose residue, inhibiting further processing Sensitive to Endoglycosidase H (Endo H) digestion Prevents removal of mannose residues on the I-3 arm of the high mannose structure Blocks activity of GlcNAc T I and -mannosidase II Sensitive to Endo H Prevents removal of mannose residues on the 1-6 arm of the high mannose structure Blocks activity of GlcNac T II (no modification of the 1-6 arm) Addition of GlcNAc, Gal and NeuAc on the 1-3 arm may occur Sensitive to Endo H

Deoxynojirimycin Castanospermine

Glucosidase I and/or II

Dependent on processing Proteins appear to be a smaller size

Deoxymannojirimycin

-mannosidase I

Proteins appear to be a smaller size

Swainsonine

-mannosidase II

Dependent on the extent of processing

Enzymes Used to Analyze Glycoproteins

Enzyme Endoglycosidase D Endoglycosidase F Endoglycosidase H -galactosidase

Type of Enzyme Endo Endo Endo Exo

Specificity Cleaves various high mannose glycans Cleaves various high mannose glycans Cleaves various high mannose glycans Removes terminal galactosides from Gal-1, 3-GlcNAc; Gal-1,4-GlcNAc; Gal1,3 GalNAc Glycoproteins between Asn and GlcNAc NeuAc-2,6-Gal; NeuAc-2,6-GlcNAc; or NeuAc-2,3-Gal

Reference 7, 11 6, 3 12, 13 5, 8, 9

Peptide:N-Glycosidase F Sialidases (Neuraminidases)

Endo Exo

10 2

This table is adapted from Molecular Biology Labfax by T.A. Brown with permission from BIOS Scientific Publishers Ltd.

References:
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