18 Number 1 November (II) 1993
`
`European
`
`publicised btwulfly
`
`i0chemistry
`
`Review in this issue
`
`Protein glycosylation
`Structural and functional aspects
`by Halina Lis and Nathan Sharon
`
`Published by Springer International
`on behalf of the
`
`Federation of European Biochemical Societies
`225 Eur.J, Biocham. ISSN 00144956 E lBCAl 218(1) 147211993) November 15. 1993
`Printld on acid has papev
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`CSL EXHIBIT 1045
`CSL v. Shire
`
`Page 1 of 31
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`European Journal of Biochemistry
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`Federation of European Biochemical Societies, 1993
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`Page20f31
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`Page 2 of 31
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` European Journal of Biochemistry
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`VOIume 218 Number1 November (Ill 1993
`
`
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`
`
`Protein chemistry and structure
`
`Sequence and expression of the gene en-
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`
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`
`
`coding the respiratory nitrous-oxide reduc-
`tase from Paracoccus denitrificans — New
`
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`
`
`and conserved structural and regulatory
`
`
`
`
`
`motifs
`
`Frank U. Hoeren, Ben C. Berks,
`
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`
`
`Stuart J. Ferguson, John E. G. McCarthy
`
`
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`Refined crystal structure of phycoerythrin
`
`
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`
`
`from Porphyrio‘ium cruentum at 0.23-nm
`
`
`
`resolution and localization of the 1' subunit
`
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`
`
`Flaif Ficner, Robert Huber
`
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`Characterization of the post—translational
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`
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`modifications in tubulin from the marginal
`band of avian erythrocytes
`
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`
`
`Manfred Riidiger, Klaus Weber
`
`
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`
`
`
`Identification of O-linked oligosaccharide
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`Chains in the activation peptides of blood
`
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`coagulation factor X — The role of the car-
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`bohydrate moieties in the activation of fac-
`tor X
`
`
`Keisuke inoue, Takashi Morita
`
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`Complete amino acid sequences of five di-
`meric and four monomeric forms of metal-
`
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`
`
`lothionein from the edible mussel Mytilus
`edulis
`
`Elaine A. Mackay, Julian Overneii,
`
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`
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`Bryan Dunbar, lan Davidson,
`Peter E. Hunziker, Jeremias H. R. Kagi,
`
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`
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`John E. Fothergili
`Recombinant coho salmon insulin-like
`
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`
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`growth factor I 7 Expression in Escherichia
`coli, purification and characterization
`
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`
`Shunsuke Moriyama,
`
`Stephen J. Duguay, J. Michael Conlon,
`
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`Cunming Duan, Walton W. Dickhoff,
`
`
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`Erika M. Plisetskaya
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`Structure and dynamics of the acidic com-
`
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`pact state of apomyoglobin by frequency-
`domain fluorometry
`
`
`Ettore Bismuto, Enrico Gratton,
`
`
`
`
`
`
`
`ivana Sirangelo, Gaetano lrace
`
`
`
`Non—cooperative effects of glucose and 2'
`
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`deoxyglucose on their metabolism in Sec-
`
`
`
`
`
`charornyces cerevisiae studied by 1H-NMFi
`
`
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`and 1aC-NMR spectroscopy
`Martina Herve, Jaime Wietzerbin,
`
`
`
`Son Tran-Dinh
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`
`
`Effect of lysine ionization on the structure
`
`
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`
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`and electrochemical behaviour of the
`
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`Met44-9Lys mutant of the blue—copper pro—
`
`
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`
`
`
`
`
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`tein azurin from Pseudomonas aeruginosa
`Mart Van De Kemp, Gerard W. Centers,
`
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`
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`
`
`Colin R. Andrew; Joann Sanders-Loehr,
`
`
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`
`
`Christopher J. Bender, Jack Peisach
`
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`
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`
`
`1727
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`29—37
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`39—48
`
`957102
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`129—141
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`143—151
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`173—181
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`1957204
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`49—57
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`103—106
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`107—116
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`153—163
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`183—194
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`205—21 1
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`2137219
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`221—228
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`229-238
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`A5
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`
`
`Contents
`
`
`
`Reviekx
`
`Protein glycosylation — Structural and func-
`
`
`
`
`
`tional aspects
`
`
`Halina Lis, Nathan Sharon
`
`
`
`
`
`
`
`
`
`Nuclic acids, protein synthesis
`
`
`
`and molecular genetics
`
`
`
`-
`
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`
`Phenotype of recombinant Leishmania do—
`
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`novani and Trypanosoma cruzi which
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`over-express trypanothione reductase —
`
`
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`Sensitivity towards agents that are
`
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`
`
`thought to induce oxidative stress
`
`
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`
`
`
`John M. Kelly, Martin C. Taylor,
`Keith Smith, Karl J. Hunter,
`
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`
`
`Alan H. Fairiamb
`
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`
`
`Increased phosphorylation of eukaryotic
`initiation factor 4;; during early activation
`
`
`
`
`
`
`
`
`
`
`of T lymphocytes correlates with in-
`creased initiation factor 4F complex for-
`
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`
`
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`mation
`
`
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`
`
`Simon J. Morley, Michaei Rau,
`John E. Kay, Virginia M. Pain
`
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`{Analysis of the DNA topoisomerase-II-me-
`
`
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`
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`d‘iated cleavage of the long terminal re-
`
`
`
`
`
`peat of Drosophiia 1731 retrotransposon
`Evelyne Nahon, Martin Best-Beipomme,
`
`
`
`Jean~Marie Saucier
`
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`Mouse gelatinase B — cDNA cloning, regu-
`
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`lation of expression and glycosylation in
`
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`¥VEHl-3 macrophages and gene organisaa
`Ion
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`Stefan Masure, Guy Nys, Pierre Fiten,
`
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`
`
`Jo Van Damme, Ghisiain Opdenakker
`
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`Qaterminants of the brain-specific expres-
`
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`
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`
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`saon of the rat aldolase C gene: ex vivo
`
`
`
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`and in vivo analysis
`
`
`
`Muriel Thomas, iman Makeh,
`Pascale Briand, Axel Kahn,
`
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`
`
`Henriette Skala
`
`
`
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`
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`N_ueleotide sequence and promoter-spe-
`
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`CIfic effect of a negative regulatory region
`
`
`
`
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`located upstream of the mouse prolifers
`
`
`
`
`
`ating cell nuclear antigen gene
`
`
`Shuhei Matsuoka,
`
`
`
`Masamitsu Yamaguchi, Yuko Hayashi,
`
`
`Akio Matsukage
`
`
`
`
`
`
`
`
`
`
`Anomalous interaction of Sp‘l and specific
`binding of an E-box-binding protein
`
`
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`
`
`
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`
`
`With the regulatory elements of the
`
`
`
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`a.K—ATPase a2 subunit gene promoter
`
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`
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`Kerko ikeda, Kei Nagano,
`Kiroshi Kawakami
`
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`
`Page 3 of31
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`Page 3 of 31
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`

`

`
`Contents (Continuation)
`
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`Partial purification and characterization of
`
`
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`
`
`a circulating hypertensive factor in sponta—
`
`
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`
`
`
`
`
`neously hypertensive rats
`Hartmut Schiiiter, Brigitte Kluth,
`
`
`
`
`
`Regina Borjesson-Stoll,
`Eckhard Nordhoff, Waiter Zidek
`
`
`
`
`Investigation of the oxygenation of phos—
`
`
`
`
`
`pholipids by the porcine leukocyte and
`
`
`
`
`
`
`human platelet arachldonate 12-lipoxy-
`
`
`
`genases
`
`Yoshitaka Takahashi,
`
`
`
`
`
`
`
`Wayne C. Glasgow, Hiroshi Suzuki,
`Yutaka Taketani, Shozo Yamamoto,
`
`
`
`
`Monika Anton, Hartmut Kilhn,
`
`
`
`
`Alan Fl. Brash
`
`
`
`
`
`
`
`Membranes and bioenergetics
`Permeability properties of peroxisomes in
`
`
`
`
`
`
`
`digitonin-permeabilized rat hepatocytes —
`Evidence for free permeability towards a
`
`
`
`
`
`
`variety of substrates
`
`
`
`Nicolette Verleur, Ronald J. A. Wanders
`
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`
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`
`
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`
`
`67—73
`
`
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`165—171
`
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`7 5—82
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`
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`Effects of glycosylation on protein strucr
`
`
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`
`
`ture and dynamics in ribonuclease B and
`
`
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`
`
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`
`
`some of its individual glycoforms
`
`Heidi C. Joao, Raymond A. Dwek
`
`
`
`
`
`
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`
`
`239*244
`
`
`
`G|y387 of murine ornithine decarboxylase
`
`
`
`
`is essential for the formation of stable ho-
`
`
`
`
`
`
`
`modimers
`
`Karin E. Tobias,
`
`
`
`Emanueile Mamroud—Kidron,
`
`Chaim Kahana
`
`
`
`
`
`
`
`245~250
`
`
`
`Enzymology
`
`
`
`
`
`
`Structure/activity relationships in porphobi-
`Iinogen oxygenase and horseradish peroxi~
`
`
`
`
`dase — An analysis using synthetic hemins
`
`
`
`
`
`
`Marcelo Fernandez, Rosalia B. Frydman,
`
`
`
`
`
`
`
`
`
`Jorge Hurst, Graciela Buldain
`
`2514259
`
`Reconstitution of holotransketolase is by a
`
`
`
`
`
`
`
`
`thiamin-diphosphate-magnesium complex
`Christine K. Booth, Peter F. Nixon
`
`
`
`
`
`
`2617265
`
`
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`
`
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`
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`The separate roles of plant cis and trans
`prenyl transferases in cisa‘l,4rpolyisoprene
`
`
`
`biosynthesis
`
`Katrina Cornish
`
`
`
`
`
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`267—271
`
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`Carbohydrates, lipids and other natural products
`
`
`
`Structure/activity relationship of leuko—
`
`
`
`triene B4 and its structural analogues in
`
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`
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`chemotactic, lysosomaI-enzyme release
`
`
`
`and receptor—binding assays
`
`
`
`
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`
`
`Olukayode Soyombo, Bernd W. Spur,
`Cecilia Soh, Tak H. Lee
`
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`59766
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`Page 4 of31
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`Molecular cell biology and metabolism
`Aminoacyl chloromethanes as tools to
`
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`study the requirements of NADPH oxidase
`
`
`
`
`
`activation in human neutrophils
`
`
`
`
`Emmanuelie Chollet-Przednowed,
`
`Florence Lederer
`
`
`Molecular analysis of chicken embryo
`
`
`
`
`
`SPARC (osteonectin)
`
`
`James A. Bassuk,
`
`
`
`
`M. Luisa lruela-Arispe, Timothy F. Lane,
`
`
`
`
`
`
`
`
`
`Janice M. Benson, Flichard A. Berg,
`E. Helene Sage
`
`
`
`Indexed in Current Contents
`
`
`
`
`
`
`
`
`
`
`83433
`
`
`
`1177127
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`
`Page 4 of 31
`
`

`

`Eur. J. Biochem . 218, 1-27 (1993)
`© FEBS 1993
`
`Review
`
`Protein glycosylation
`Structural and functional aspects
`
`Halina LIS and Nathan SHARON
`Deparunent of Membrane Research and Biophys ics, The We izmann lnstitute of Science. Rehovot, Israel
`
`(Received April 16/July 15, 1993) - EJB 93 0558/0
`
`During the Jast decade, there have been enormous advances in o ur knowledge of glycoproteins
`and the stage has b'een set for the bio technological production of many of them for therapeutic use.
`These advances are reviewed, with special emphasis on the structure and function of the glycopro(cid:173)
`teins (excluding the proteoglycans). Current methods for structural analysis o f g lycoproteins are
`surveyed, as are nove] carbohydrate - peptide linking groups, and mono- and o ligo-saccharide con(cid:173)
`stituents found in these macromolecules. The possible roles of the carbohydrate units in modulating
`the physicochemical and biological properties of the parent proteins are discussed, and evidence is
`presented on their roles as recognitio n determinants between molecules and cells, or cell and cells.
`Finally, examples are given of changes that occur in the carbohydrates of soluble and cell-surface
`glycoproteins during differentiation, growth and malignancy, which further high light the important
`role of these substances in health and disease.
`
`Among the different types of covalent modifications that
`newly synthesized proteins undergo in living organisms,
`none is as common as glycosylation (1 - 6]. It is also the
`most diverse, both with respect to the kinds of amino acid
`that are modified and the structures attached. The origins for
`this diversity are chemical as well as biological. The former
`results from the ability o f monosaccharides to combine with
`each other in a variety of ways that differ not only in se(cid:173)
`quence and chain length, but also in ano mery (a or {J), posi(cid:173)
`tion of linkages and branching po ints. Further structural di(cid:173)
`versification may occur by covalent attachment of sulfate,
`phosphate, acetyl or methyl groups to the sugars. Therefore,
`in theory, an enormo us variety of glycans, both oligosaccha(cid:173)
`rides and polysaccharides, can be generated from a relatively
`limited number of monosaccharides. Biological diversity de(cid:173)
`rives from the fact that, whereas proteins are primary gene
`products, g lycans are secondary gene products. As a result,
`glycosylation is species- and cell-specific, and is determined
`as well by the structure of the protein backbone and the car(cid:173)
`bohydrate attachment site. This means that glycosylation of
`any protein is dependent on the cell or tissue in which it is
`produced and that the po lypeptide encodes informatio n that
`directs its own pattern of g lycosylation.
`ln an individual g lycoprotein more than one carbo hyd rate
`unit is often present, attached at different positions by either
`an N-linkage, an 0 -linkage or both . Moreover, each attach-
`
`to N. Sharon, Depan ment of Membrane
`Correspondence
`Research and Bio physics, The Weizmann lnstitute of Science,
`IL-76100 Rehovot, Israel
`Abbreviations. C HO, Chinese hamster ovary; GIPtdlns, glyco(cid:173)
`syl-phosphatidylinositol; GM-CSF g ranulocyte-macrophage colon y
`stimulating facto r ; hCG, human c horio nic gonado tropin ; LH, Iuto(cid:173)
`tropin ; N-CAM, neural cell adhesion molecule; tPA, tissue plasmin(cid:173)
`ogen activator.
`
`ment site frequently accommodates different glycans, a phe(cid:173)
`nomeno n referred to as site heterogeneity. This resul ts in
`microheterogeneity of the whole molecule and creates
`discrete subsets, or g lycoforms, o f a glycoprotein that have
`different physical and biochem ical properties, which, in tum,
`may lead to functional di versity [7]. ln short, glycosylation
`of a polypeptide usually generates a set of glycoforms, ail o f
`which share an identical backbone but are dissimilar either
`in the structure or disposi tio n of their carbohydrate units or
`in both. The earlier view that heterogeneity o f the carbohy(cid:173)
`drates of glycoproteins is random, mainly d ue to the lack of
`fidelity in their synthesis, seems no longer tenable, since the
`relative proportions o f such glycoforms appear to be repro(cid:173)
`ducible and highly regulated. They depend on the cellular
`environment in which the protein is g lycosylated and may
`therefore vary with the type, as well as the physio logical
`state, of the o rganism, tissue or cell in which the glycoprotein
`is made.
`The ubiquity of glycosylation is well established. It oc(cid:173)
`curs without exception in integral membrane proteins of
`higher organisms and is quite commo n w ith secretory pro(cid:173)
`teins. For instance, in blood serum, almost all proteins are
`g lycosylated, as are those in hen egg white. Glycoproteins
`are now known to occur also in the cytoplasm and nucleus
`[8]. Whereas bacteria were for a long time considered to lack
`the ability to synthesize glycoproteins, this now appears not
`to be the case. Many species of archaebacteria, as well as of
`eubacteria, produce glycoproteins, although mostly of types
`not found in other organisms (9-11 ).
`During the last decade, there has been a vast expansion
`in our knowledge of the distribution of g lycopro teins in na(cid:173)
`ture, and of their structure, biochemistry and biosynthesis;
`important insights have also been obtained into their roles.
`Detection and isolation of g lycoproteins have been facili-
`
`Page 5 of 31
`
`

`

`2
`
`1982
`
`GlcNAc
`N
`
`Asn
`
`GalNAc Gal
`Man
`Xyl
`0
`0
`
`L-Fuc Gal
`0
`0
`
`L-Ara
`Gal
`0
`
`COOH
`
`1992
`
`GalNAc
`Glc
`GlcNAc
`L-Rha
`N
`
`GalNAc Gal
`GlcNAc Glc
`Xyl
`Man
`0
`0
`
`L-Fuc Gal
`0
`0
`
`L-Ara
`Gal Glc
`0
`0
`
`H2N
`Fig. 1. Protein-carbohydrate linkages known in 1982 and in
`1992. GPI stands for glycosyl-phosphatidylinositol.
`
`CO-GPI
`
`tated, not the least thanks to the availability of an increasing
`range of lectins with a wide spectrum of specificities which
`are capable of distinguishing subtle differences in the struc(cid:173)
`ture of oligosaccharide units of glycoproteins (12- 14). Re(cid:173)
`finements of known separation and analytical methods, as
`well as introduction of new technologies, have made it pos(cid:173)
`sible to determine complex glycan structures at the nano(cid:173)
`mole, and sometimes even picomole, level in relatively short
`periods of time. As a consequence, the number of known
`structures of carbohydrate units of glycoproteins has grown
`immensely and the early assumption, that living organisms
`form .only an exceedingly small fraction of the theoreticaJly
`possible molecular permutations of the dozen or so monosac(cid:173)
`charides typically found in glycoconjugates, seems no longer
`justified. Not only have nove! structures been discovered,
`but so too have new monosaccharide constituents and new
`linkages between the peptide backbone and the carbohydrate
`unit. The latter point is illustrated in Fig. 1 which compares
`the linkages known today with those known a decade earlier.
`The nove! linkages include, in addition to hitherto unknown
`N- and 0-glycosidic bonds, the glycosyl-phosphatidylinositol
`(G!Ptdlns) anchor, a new class of widely occurring linkage,
`where the carbohydrate is attached to the C-terminal amino
`acid of the protein via ethanolamine phosphate (1 5-18]. It
`should be noted, however, that this kind of attachment of
`carbohydrate to the protein is not a glycosylation process in
`the strict sense, since the sugar is not bound to the polypep(cid:173)
`tide chain by a glycosidic linkage ; it has been termed 'glypi(cid:173)
`ation'.
`Striking advances have been made in synthetic carbohy(cid:173)
`drate chemistry. Linear or branched oligosaccharides con(cid:173)
`sisting of up to a dozen units, as well as different glycopep(cid:173)
`tides, can now be produced in the laboratory ; these include
`constituents of N- and 0-glycoproteins and of the GIPtdlns
`anchor (1 9, 20]. Simple procedures for enzymatic synthesis
`of oligosaccharides, at a hundred milligram scale using im(cid:173)
`mobilized enzymes, have also become available [21, 22].
`Nevertheless, synthesis of most oligosaccharides found in
`glycoproteins is still difficult (or impossible), as is the scaling
`up of the synthetic procedu res to the gram Jevel. The syn(cid:173)
`thetic products are widely employed as reference com(cid:173)
`pounds, for the investigation of specificity and structure/
`function relationships of enzymes, lectins, antibodies, etc.
`Their application as potential drugs, e.g. for prevention of
`microbial infections or inflammation, is under intensive in(cid:173)
`vestigation. Conjugation of oligosaccharides of known struc-
`
`ture to proteins (e.g. bovine serum albumin) affords ' neogly(cid:173)
`coproteins' with desirable carbohydrate units [23]. These
`compounds too are useful for probing the specificity of car(cid:173)
`bohydrate-binding proteins and as affinity matrices for the
`isolation of such proteins. In addition, they serve as imrnuno(cid:173)
`gens for the production of antibodies against oligosaccha(cid:173)
`rides and in studies of the role of the carbohydrate in glyco(cid:173)
`proteins.
`Progress has been made in our knowledge of the three(cid:173)
`dimensional structures of oligosaccharides, both free and
`protein-linked, based on nuclear magnetic resonance (NMR),
`various modelling techniques and X-ray crystallography
`[24 - 30]. lt has become apparent that, in solution, the oligo(cid:173)
`saccharides are flexible molecules that can adopt different
`conformations, only a few of which are recognized by carbo(cid:173)
`hydrate-specific proteins.
`The principal biosynthetic pathways leading to the pro(cid:173)
`duction of mature glycoproteins by glycosyltransferases, gly(cid:173)
`cosidases and carbohydrate-modifying enzymes, and in par(cid:173)
`ticular the fi ne detail s of the dolichol phosphate cycle, in
`which the Glc3Man 9(GlcNAc)2 precursor of the commonly
`is synthesized , have been
`occurring N-oligosaccharides
`known for some time [31]. White much attention is still be(cid:173)
`ing given to purification and characterization of the enzymes
`involved, and to the reactions they catalyze, emphasis has
`shifted to topological aspects, control mechanisms and mo(cid:173)
`lecular biology of glycosylation. Under intense investigation
`are problems such as subcellular sites of glycosylation, trans(cid:173)
`location of sugars from the cytoplasmic face to the lumen of
`the endoplasmic reticulum and the Golgi apparatus, traffick(cid:173)
`ing between organelles (e.g. from the Golgi to lysosomes)
`and, most importantly, regulation of glycoprotein processing
`and maturation. Much of our knowledge in these areas has
`been obtained with the aid of mutant mammalian cell lines,
`selected mostly by virtue of the ir resistance to the toxic ac(cid:173)
`tion of lectins and shown to be deficient in certain enzymes
`involved in individual steps of protein glycosylation [32].
`Another source of information cornes from the use of specific
`inhibitors of transferases (e.g. tunicamycin) and of glycosi(cid:173)
`dases (such as castanospermine, nojirimycin and swain(cid:173)
`sonine) [33, 34]. Mapping of the subcellar sites of protein
`glycosylation is aided by the use of lectins and of antibodies
`to purified glycosylated enzymes [35, 36].
`New approaches became available with the emergence of
`genetic engineering techniques. For instance, oligonucleo(cid:173)
`tide-directed mutagenesis allows for specific changes in the
`primary structure of glycoprote ins and faci litates the exami(cid:173)
`nation of factors governing site-speci tïc glycosylation and
`oligosaccharide processing. ln glycoproteins with more than
`one glycan, mutagenesis provides insights into the contribu(cid:173)
`tion of each glycan to the overall properties of the molecule.
`Evidence has accumulated for the existence of proteins
`which mediate the transport of sugar nucleotides across the
`membranes of the endoplasmic reticulum and the Golgi ap(cid:173)
`paratus [37, 38]. The transporters, or antiporters, facilitate the
`entry of the sugar donor into the lumen of these organelles in
`a reaction coupled to the equimolar exit of the corresponding
`nucleoside monophosphate. Sorne of the transporters have
`been partially purified and shown to be both organelle- and
`substrate-specific.
`The role of the Golgi complex in the ordered remodelling
`of N-oligosaccharide chains and the biosynthesis of 0 -gly(cid:173)
`cans is firml y established [35, 37]. lt has also become clear
`that this organelle consists of a series of functionally distinct
`compartments: cis, media! and trans. As glycoproteins pass
`
`Page 6 of 31
`
`

`

`through these compartments, they acquire their 0 -units,
`while the N-oligosaccharides, the precursor of which is at(cid:173)
`tacbed to the growing polypeptide chain in the endoplasmic
`reticulum, undergo a series of sequential trimming and elon(cid:173)
`gation reactions, as if on an assembly line [36). The genes
`coding for the relevant enzymes are being cloned and se(cid:173)
`quenced at an increasingly fast rate; studies on their regula(cid:173)
`tion are in progress [39-42). A recent milestone is the clon(cid:173)
`ing and sequencing of the cDNAs which code for the glyco(cid:173)
`syltransferases that determine human blood types A, and B
`( a-1,3-N-acety lgalactosam i ny ltransferase and a-1,3-galacto(cid:173)
`syltransferase, respectively), and of the corresponding cDNA
`from cells of the H(O) type [43). The deduced sequences for
`the two transferases differ only in four arnino acid residues,
`while a critical single base deletion found in the O individ(cid:173)
`uals is predicted to give rise to an entirely different protein
`which would be expected to be nonfunctional.
`Severa! glycosyltransferases have been shown Lo exhibit
`branch specificity, which accounts for the marked differences
`in chain length sometimes fourid between different branches
`of the same glycan f 44). An insight into the mechanism by
`whicb the protein backbone may control glycosylation was
`provided by the finding that, in addition to the combining
`site(s) for the sugar donor and acceptor, glycosyltransferases
`can contain a site that recognizes certain features in the pep(cid:173)
`tide moiety of the acceptor glycoprotein [45).
`Recent work has clarified several aspects of the catabo(cid:173)
`lism of N-glycoproteins [46). ln this process, a series of lyso(cid:173)
`somal enzymes act in a highly ordered manner to ensure the
`complete degradation of glycoproteins. lt is achieved by step(cid:173)
`wise hydrolysis of the major portion of their glycans by a set
`of exo-glycosidases, followed by the disassembly of the pro(cid:173)
`tein and the carbohydrate - peptide linkage region. An alter(cid:173)
`native pathway for the degradation of glycoproteins starts
`with proteolysis of the polypeptide backbone and involves
`the participation of specific endoglycosidase(s) [47, 48). The
`physiologica1 importance of high precision in the lysosomal
`degradative system is clearly illustrated by the occurrence of
`serious, often fatal, disorders in indi viduals with genetic de(cid:173)
`fects in glycosidase production [49, 50]. Details of the degra(cid:173)
`dation of 0 -glycans are largely unknown but, as recently
`shown, in this case, too, genetic defects in glycosidase pro(cid:173)
`duction may lead to serious disorders (51].
`The fact that the carbohydrate units of glycoproteins have
`been conserved in evolution and the growing awareness of
`the widespread occurrence and structural diversity of glyco(cid:173)
`proteins, coupled with the realization that oligosaccharide
`Structures of glycoproteins sometimes undergo drarnatic
`~hanges with differentiation and in pathological processes,
`mtensified the search for their biological role(s). The ability
`~f the carbohydrate groups to modulate the physical proper(cid:173)
`ties of the protein to which they are attached, especially the
`overall folding of the nascent polypeptide chain, as well as
`to protect it against proteolysis, is well documented [1 -4].
`More importantly, there is increasing evidence for the con(cid:173)
`Cept, formulated over 20 years ago [52], that carbohydrates
`act as recognition deterrninants in a variety of physiological
`llnd pathological processes (7, 53 - 57]. These include clear(cid:173)
`ance of glycoproteins from the circulatory system [52], intra(cid:173)
`Cellular trafficking of enzymes [58] and a wide range of
`Cell-cell interactions, from the attachment of sperm to ova
`(59], to adhesion of infectious microorganisms to host tissues
`(60-62]. Particularly exciting is the recent demonstration
`th~t binding of carbohydrates on the surface of leukocytes,
`\\'Jth a class of animal lectins designated 'selectins', controls
`
`3
`
`Table 1. Sorne glycosylated proteins of therapeutic interest. T his
`table is based largely on the review of Rasmussen [67].
`
`Glycoprotein
`
`Carbohy- Require- Biotech- Clinical
`drate-
`nology
`ment of
`use
`peptide
`carbohy-
`pro-
`linkage
`drate for duction
`activiry
`
`\
`\
`
`1
`
`\
`
`+
`:.t
`+
`
`+
`
`+
`
`+
`
`+
`+
`
`+
`
`+
`
`+
`+
`
`+
`
`+
`
`+
`
`+
`+
`
`+
`
`:.t
`
`+
`
`+
`
`:.t
`
`N and 0
`N
`
`0
`
`a,-Antitrypsi n
`N
`Coagulation factor VIII N and 0
`Erythropoietin
`N and 0
`Follicle stimulating
`hormone
`Glucocerebrosidase
`Granulocyte colony-
`stimul ating factor
`Granulocyte-macro-
`phage colony-
`stimulating factor
`Human chorionic
`gonadotropin
`Interleukin-2
`lnterferon-P
`Interferon-y
`Protein C
`Soluble CD4
`Tissue plasminogen
`activator
`
`N and 0
`
`N and 0
`0
`N
`N
`N
`N
`
`N
`
`leukocyte traffic by mediating adhesion of these cells to re(cid:173)
`stricted portions of the endothelium and their recruitment to
`inflammatory sites (63-65). Within a short period of time,
`the study of selectins and their receptors has become, per(cid:173)
`haps, the most active area in glycobiology. Intensive attempts
`are in progress to design carbohydrate-based selectin inhibi(cid:173)
`tors, which, in tum, may be candidates for a new class of
`anti-inflammatory drugs (66].
`This is one example of bow increased knowledge of car(cid:173)
`bohydrate structure and fonction might be utilized for thera(cid:173)
`peutic purposes. Another example is the enzymatic modifica(cid:173)
`tion of the glycan of the enzyme glucocerebrosidase (gluco(cid:173)
`sylcerarnidase), which is essential for its clinical use (under
`the tracte name Ceredase) in the treatment of patients with
`Gaucher' s disease [68, 69]. Tt is the first, and thus far prob(cid:173)
`ably the only, case of enzyme replacement therapy, a concept
`suggested some 30 years ago. Also, genetic engineering
`makes it possible to produce glycoproteins in heterologous
`systems on a large scale, both for research purposes and for
`therapeutic use (Table 1). We are indeed witnessing the emer(cid:173)
`gence of glycotechnology [70] , a branch of biotechnology
`that uses nove! approaches to manipulate carbohydrates or
`related materials, with the aim of creating new products or
`new procedures for the betterment of our lives. An impres(cid:173)
`sive example is erythropoietin, a circulating glycoprotein
`hormone that stimulates erythropoiesis, which has the dis(cid:173)
`tinction of being the first recombinant glycoprotein produced
`industrially for