GLYCOPROTEINS


New Comprehensive Biochemistry

Volume 29a

General Editors

A. NEUBERGER
Loridovi

L.L.M. van DEENEN
Utrwht

ELSEVIER
Amsterdam - Lausanne - New York - Oxford - Shannon - Tokyo


Glycoproteins

Editors

J. Montreuil
Uniivrsitc' ties Sciences ct Technologies de Lille.
Lahoratoiw dc Cliiniie Biologiyire.
( U M R no 111 dir CNRS).59655 Villeneuiv d'Asy Cede.1.. Fi-ance

J.F.G. Vliegenthart
Bijivet Center f i l l . Bioniolecular- Reseal-cli.

Department of'Bio-or;qanic~Cheniistry,
P.O. Bo.v 80.075. 3508 TB Utiuclit, The Netherlands

H. Schachter
Department of' Biochenli.yt1.y Resear-ch, Hospitalfi)r-Sick Child.en,
555 Uniiwsity Aivnire, Toianto. Ont. M5G 1x8.Canada

Amsterdam

-

1995
ELSEVIER
Lausanne - New York - Oxford

-

Shannon - Tokyo


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V

Preface
The presence of sugar moieties in proteins had been the subject both of speculation and
experiment for some time when Pavy (1893) carried out experiments with coagulated
egg-white, and drew the conclusion that this mixture of proteins and many other
proteins contained carbohydrate in a covalent combination. Several other workers
confirmed the somewhat messy experiments of Pavy, but most workers in the field
believed that apart from mucins and mucoids, ordinary proteins consisted only of
amino acid residues and that the presence of sugars in hydrolysates was due to impurities such as mucoids. Such belief was expressed by Plimmer (1917) and by Levene
(1923) in textbooks which carried considerable prestige at the time that they were

published.
This was the situation that I met in 1936 when, after obtaining my Ph.D. in
Biochemistry, I became interested in this problem. Almost all crystalline proteins
known at that time were free of sugar with the possible exception of egg albumin. It
was important therefore to find out whether egg albumin really contained covalentlybound sugar residues. As a first step I tried to separate the carbohydrate by physical
means, such as prolonged dialysis, repeated ultrafiltration and crystallization. It was
impossible to separate carbohydrate from the protein by any of the methods including
mild chemical techniques. I will not discuss the steps taken then to hydrolyse all the
peptide linkages whilst leaving the carbohydrate content intact, but I believe most of
the conclusions drawn at that time were essentially correct. The first conclusion was
that the carbohydrate is almost certainly combined with the peptide moiety by a
covalent bond. The second conclusion was that the carbohydrate is present as one unit,
as shown by the molecular weight estimations. A third conclusion which was implied
was that the sequence of sugar residues in a glycopeptide is as constant as it is in the
peptide moiety. This assumption turned out to be erroneous. At that time it would have
been difficult to identify the amino acid linking the peptide to the carbohydrate
component. It was a few years before paper chromatography was developed and the
amino acid concerned, i.e. asparagine, gave no specific reactions. After my paper was
published (1938) I spent some time trying to identify the linking amino acid. Two of
the nitrogen atoms of the complex were associated with the missing linkage and I
believed that either asparagine or glutamine might be involved. Indeed, I did isolate a
small amount of aspartic acid from the hydrolysate of the complex but the quantity
appeared to be too small to justify a definite conclusion. Within a year of the publication
of my paper the Second World War had started and no long-term research was felt to
be appropriate.
The problem of the linkage of egg albumin was taken up again by my colleagues
and myself in 1956 and led to a paper which appeared in Nurure in 1958. In this paper
it was shown that asparagine was the linking compound. Experiments similar to our
own were also described at about the same time by Cunningham and his colleagues and



VI

by Jevons. More conclusive findings were reported later by ourselves and the N-(L-Paspartyl)-~-D-glucopyranosy~amine
was later synthesized with G.S. Marks. The complete identification of synthetic material with the natural glycopeptide finally clinched
the structure. This type of linkage is probably the most widespread in glycoproteins.
Since this work was done in the early 1960s, the number of proteins shown to have
sugar residues covalently bound to the peptide structure has increased enormously.
There have been other linkages demonstrated in many glycoproteins such as the linkage
between galactose and serine or threonine. They are present in plants, bacteria, animals,
and even in archaebacteria, and most probably it is the largest group of proteins. In
recent years we have come to appreciate the fact that the glycosylation of peptides is
only one, but perhaps the most widespread, example of post-translational change of
protein synthesis. We can in fact divide natural products into two categories. One
category represents the proteins which are controlled essentially by the sequence of
bases in DNA. Any change in the sequence of bases in DNA is a mutation, and the
whole process of protein synthesis tries to reduce the occurrence of mutations to a
minimum. Glycosylation of peptides, like the hydroxylation of proline and glycine
residues which occur in collagen, or the iodination of tyrosine residues which occurs
in thyroglobulin, takes place either during the translation process of protein synthesis,
or immediately afterwards. They are controlled by enzymes which themselves are
products of protein synthesis. However, these non-translational processes are not
subject to the same fidelity as amino acid sequence in peptides. They depend on
specificity of enzymes, their location in the cell, their activity, and many other factors,
which must show considerable variability. In other words, we do not expect the same
constancy i n composition or in structure as we would with peptides. Thus, it is likely
that fatty acid residues in phospholipid might vary considerably even within one organ.
Similarly, the detailed structure of the carbohydrate component in the glycopeptide
might vary within certain prescribed limits. This accounts for the fact that in most, and
possibly all, glycoproteins there is variability in the polysaccharide structure, while in

the peptide moiety sequence is constant. This is the case even in the relatively simple
case of egg albumin.
The last problems that I want to discuss concern the functions of the sugar residues
in glycoproteins. The complex type of biosynthesis of the carbohydrate component of
glycoproteins is truly amazing, and it is very difficult to see at present how this
sequence of enzymatic reactions involving controlled addition and deletion of sugars
to and from the glycopeptide is regulated. It is thus reasonable to believe that this
complicated mechanism serves an important biological function. It is likely there is still
much to be discovered in this field, but some general points can be made. The addition
of carbohydrate to peptide structure will change the shape and size of the protein
molecule. This is likely to affect the access of proteolytic enzymes, and it will almost
certainly influence such factors as heat stability, solubility and many physical and
chemical properties. It is also likely to affect interaction with other proteins or non-protein components of the cell, and also affect interaction with other cells. It is almost
certain that glycoproteins are prominently involved with social behaviour of cells. It is
thus not surprising that the lifetime of a protein in a whole organism is greatly affected
by sugar residues present in the glycoprotein in question. In the case of a hormone, it


VII

is likely that whilst activity in vitro may not be changed by the possession of a
carbohydrate group, its survival in the whole organism might be greatly modified by
the sugar component of the glycoprotein.
I wish to say finally that this book contains authoritative reviews on many aspects
of our present state of knowledge of glycoproteins. In this vastly expanding field, no
review is likely to be exhaustive or up-to-date in a few years’ time. However, I hope it
will be helpful to most readers and will stimulate them to further work in this important
field of biochemistry.
Professor Albert Neuberger
Charing Cross and Westminster Medical School

Department of Biochemistry
Fulham Palace Road
London W 6 SRF, UK


Laurens van Deenen f


In Memoriam

To our great sorrow we have to report the death of Professor
Laurens van Deenen, on September 4,1994.
Laurens van Deenen was an outstanding scientist who
made great contributions to our knowledge of the structure
and function of lipids of biological importance, and their interaction with other components of the cell. He also increased our
understanding of the nature of membranes in biological systems and the relevant fields of enzymology. He was a pioneer
full of original ideas, and canied out his work with the best
available methods, always being cautious in the interpretation of his results.
Laurens van Deenen was the creator of a school which was
internationally recognized and as a person was generous to his
co-workers. He had wide interests outside his own discipline.
He joined the Editorial Board of Biochimica et Biophysica
Acta, and was one of the Managing Editors from 1964-1993
and Chairman during the years 1983-1989.
From 1977 onwards, Laurens van Deenen was also involved
in the editorship of the original comprehensive Biochemistry
series, and was one of the initiators in developing a second
series, New Comprehensive Biochemistry, being responsible
for the realization of this venture. Here his wide knowledge of
biochemistry, stretching far beyond his own fields, his wisdom

and his judgement were great assets.
Throughout his life, Laurens van Deenen set high standards
for his own work; he was a stimulating colleague and a good
friend.
London, October 1994

Albert Neuberger


x

List of contributors
Friedrich Altmann
Institut fur Chemie, Universitatfur Bodenkultur, Gregor-Mendelstrasse 33, A-I 180
Vienna, Austria

T. Bielfeldt
Institute of Organic Chemistry, University of Hamburg, Martin-Luther-King-Platz 6,
20146 Hamburg, Germany
Malgorzata Bielinska
Department of Molecular Biology and Pharmacology and Obstetrics and
Gynecology, Washington University School of Medicine, 660 S. Euclid Avenue, St.
Louis, MO 63110, USA
Irving Boime
Department of Molecular Biology and Pharmacology and Obstetrics and
Gynecology, Washington University School of Medicine, 660 S. Euclid Avenue, St.
Louis, M O 63110, USA
Inka Brockhausen
Department of Biochemistry Research, The Hospital for Sick Children, 555
University Avenue, Toronto M5G 1x8,and Biochemistry Department, University of

Toronto, Toronto M5S 1A8, Ontario, Canada
Christi an Cambillau
Laboratoire de Cristallographie et de Cristallisation des Macromol&cules
Biologiyues, CNRS URA 1296, Faculte' de Me'decine Secteur-Nord, Bd. Pierre
Dramard, 13916 Marseille Cedex 20, France
Raymond T. Camphausen
Small Molecule Drug Discovery, Genetics Institute, 87 Cambridge Park Drive,
Cambridge, MA 02140, USA
Dale A. Cumming
Small Molecule Drug Discovery, Genetics Institute, 87 Cambridge Park Drive,
Cambridge, MA 02140, USA
Alan D. Elbein
Department of Biochemistry and Molecular Biology, The University of Arkansas for
Medical Sciences, Little Rock, AR, USA


XI

Mary Catherine Glick
The Children Is Hospital of Philadelphia, Department of Pediatrics, University of
Pennsylvania School of Medicine, 34th Street and Civic Center Boulevard,
Philadelphia, PA 19104, USA
Frank W. Hemming
Department of Biochemistry, University of Nottingham, Queen's Medical Centre,
Nottingham NG7 2UH, UK

S.W. Homans
University of Dundee, Department of Biochemistry, Medical Sciences Institute,
Carbohydrate Research Centre, Dundee DD1 4HN, UK
Billy G. Hudson

Department of Biochemistry and Molecular Biology, University of Kansas Medical
Center, Kansas City, KS 66160-7421, USA
Frans M. Klis
Institute of Molecular Cell Biology, University of Amsterdam, BioCentrum
Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands
Viktoria Kubelka
1nstitut.fur Chemie, Universitat f u r Bodenkultur, Gregor-Mendelstrasse 33, A-I 180
Vienna, Austria

L. Lehle
Lehrstuhl f u r Zellbiologie und Pjlanzenphysiologie, Universitut Regensburg, 93040
Regensburg, Germany
Leopold Marz
Institut f u r Chemie, Universitatfur Bodenkultur, Gregor-Mendelstrasse 33, A - I I80
Vienna, Austria
Jean Montreuil
Universite' des Sciences et Technologies de Lille, Laboratoire de Chimie Biologique,
(UMR no I I 1 du CNRS), 59655 Villeneuve d'Asq Cedex, France
Milton E. Noelken
Department of Biochemistry and Molecular Biology, University of Kansas Medical
Center, Kansas City, KS 66160-7421, USA
Y.T. Pan
Department of Biochemistry and Molecular Biology, The University of Arkansas for
Medical Sciences, Little Rock, AR, USA
H. Paulsen
Institute of Organic Chemistry, University of Hamburg, Martin-Luther-King-Platz 6,
20146 Hamburg, Germany


XI1


S. Peters
Institute of Organic Chemistry, University of Hamburg, Martin-Luther-King-Plat2 6,
20146 Hamburg, Germany
Jiirgen Roth
Division of Cell and Molecular Pathology, Department of Pathology, University of
Zurich, Schmelzbergstr. 12, CH-8091 Zurich, Switzerland
I

Harry Schachter
Department of Biochemistry Research, Hospital for Sick Children, 555 University
Avenue, Toronto, Ont. M5G 1x8, Canada
Erika Staudacher
Institut fur Chemie, Universitatfur Bodenkultur, Gregor-Mendelstrasse 33, A-1180
Vienna, Austria
Arnd Sturm
Friedrich Miescher-lnstitut, Postfach 2543, CH-4002 Basel, Switzerland
Manfred Sumper
Universitat Regensburg, Lehrstuhl Biochemie I, Postjiach 397, 93053 Regensburg,
Germany
W. Tanner
Lehrstuhl fur Zellbiologie und Pflanzenphysiologie, Universitat Regensburg, 93040
Regensburg, Germany
Andre Verbert
Laboratoire de Chimie Biologique, UMR du CNRS No. I I I, Universite'des Sciences
et Technologies de Lille, 59655 Villeneuve d'Ascq, France
Johannes F.G. Vliegenthart
Bijvoet Center for Biomolecular Research, Department of Bio-organic Chemistry,
P.O. Box 80.075, 3508 TB Utrecht, the Netherlands
Winifred M. Watkins

Department of Haematology, Royal Postgraduate Medical School, Hammersmith
Hospital, Du Cane Road, London W12 ONN, UK
Felix T. Wieland
lnstitut fur Biochemie I, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany
Hsiang-ai Yu
Small Molecule Drug Discovery, Genetics Institute, 87 Cambridge Park Drive,
Cambridge, MA 02140, USA


XI11

Contents
...............................................

V

List of contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

X

Chapter 1 . The history of glycoprotein research. a personal view
Jean Montreuil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

Preface

Evolution and revolutions of ideas in the field of glycoproteins . An overview . . . . . . .
The early history: the birth of glycoprotein biochemistry . . . . . . . . . . . . . . . . . .
The years 1967-1969 and the birth of the molecular biology of glycoconjugates . . . . . .

3.1.
Two discoveries change the face of the world of glycoconjugates . . . . . . . . .
3.1.1. Glycans are recognition signals for membrane lectins . . . . . . . . . . .
3.1.2. Glycans are modified in cancer cell membranes . . . . . . . . . . . . . . .
3.2.
Three-dimensional structure of glycans. From speculation to reality . . . . . . . .
3.2. 1. The first images: speculation . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2. The modern views: reality . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1
3
7
7
7
7
8
8
9
10
10

Chapter 2 . Primary structure of glycoprotein glycans
Johannes F .G. Vliegenthart and Jean Montreuil . . . . . . . . . . . . . . . . . . . . . . . . . .

13

I.


2.
3.

1 . 1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.2. Intact glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3. Partial structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.4. Structure determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Monosaccharide constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. The glycan protein linkages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. The inner-core and antenna concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Classification and nomenclature of glycans and glycoproteins . . . . . . . . . . . . . .
2.5. Microheterogeneity of glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6. Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13
13
14
16
17
17
20
. 21
23
24
24
25

25


XIV
Chapter 3. 3 0 Structure . I . The structural features of protein-carbohydrate interactions revealed
by X-ray crystallography
Christian Cambillau . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
29
Proteinxarbohydrate complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.1,
Periplasmic receptor proteins: complexes with mono- and disaccharides . . . . . . 3 1
2 . I . 1 . The arabinose binding protein (ABP) . . . . . . . . . . . . . . . . . . . . 31
2 . I .2. The galactose/glucose binding protein (GBP) . . . . . . . . . . . . . . . . 31
2 . I .3. The maltose binding protein (MBP) . . . . . . . . . . . . . . . . . . . . . 31
35
2.2.
Vegetal lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1. Lathyrus ochrus isolectins . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.2.2. Pea lectin and Concanavalin A . . . . . . . . . . . . . . . . . . . . . . . .
43
2.2.3. Griffoorzia sirnplicifolia lectin . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.2.4. Erythrina corallorlendron lectin . . . . . . . . . . . . . . . . . . . . . . . 43
2.2.5. Wheat germ agglutinin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.3.
Animal lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
2.3.1. C-type lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
2.3.2. S-type lectins (galectins) . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
2.4. Fab antibody fragments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

2.5.
Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
2.5.1. Lysozyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
2.5.2. Glycogen phosphorylase b . . . . . . . . . . . . . . . . . . . . . . . . . .
55
2.5.3. Sialidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
59
2.5.4. a-Amylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 . Structural rules for establishing protein-carbohydrateinteractions . . . . . . . . . . . . . 59
3.1.
Hydrogen bonds and ordered water molecules . . . . . . . . . . . . . . . . . . . . 59
3.2.
Hydrophobic stacking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.3.
Saccharide conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
4 . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
61
61
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
63
1.

2.

Chapter 3. 3D Structure . 2. Three dimensional structure of oligosaccharides explored by NMR
nncl computer calculations
S.W. Hornans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
67
1.


2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I .I .
Why study oligosaccharide conformation and dynamics? . .
1.2
Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.
Solution conformations of oligosaccharides . . . . . . . . . . . . .
2.1,
Conformational parameters of oligosaccharides . . . . . . .
2.2.
Theoretical predictions . . . . . . . . . . . . . . . . . . . .
2.2.1. Which forcefield? . . . . . . . . . . . . . . . . ..
2.2.2. Energy minimisation . . . . . . . . . . . . . . . ..
2.2.3. Gridsearch calculations . . . . . . . . . . . . . . .

. . . . . . . . . . . . 67

. . . . . . . . . . . . 67
. . . . . . . . . . . .67
. . . . . . . . . . . . 68
. . . . . . . . . . . .68
. . . . . . . . . . . . 68

. . . . . . . . . . . .68
. . . . . . . . . . . .69
. . . . . . . . . . . .70



xv
2.2.4. Simulated annealing . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
71
72
2.2.5. Molecular dynamics simulations . . . . . . . . . . . . . . . . . . . . . . .
2.2.6. Calculations in vacuo vs . explicit inclusion of solvent . . . . . . . . . . . 72
2.3.
Nuclear magnetic resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
2.3. I . Nuclear Overhauser effects . . . . . . . . . . . . . . . . . . . . . . . . . 73
2.3.2. Spin-spin coupling constants . . . . . . . . . . . . . . . . . . . . . . . . 74
3. Recentresults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
77
Are oligosaccharides ‘rigid’ or ‘flexible’? . . . . . . . . . . . . . . . . . . . . . .
3.1.
3.1.1. Computation of theoretical NOEs from gridsearch calculations . . . . . . 77
3.1.2. Computation of theoretical NOEs from molecular dynamics simulations . 77
3.1.3. Results from spin-spin coupling constant data . . . . . . . . . . . . . . . 77
3.1.4. Relaxation-time measurements . . . . . . . . . . . . . . . . . . . . . . .
78
80
3.2.
Specific examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1. N-linked glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
80
3.2.2. Blood group oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . 81
82
3.2.3. Glycolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

83
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
84
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
84

Chapter 4. Chemical synthesis of glycopeptides
H . Paulsen. S. Peters and T. Bielfeldt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

87

1.
2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Synthesis of 0-glycopeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Synthesis of 0-glycopeptides with 2-acetamido-2-deoxy-a-D-galacto-pyranosyl
2.1 .
linkage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
2.1. 1 . Glycopeptide synthesis in solution . . . . . . . . . . . . . . . . . . . . . .
88
2.1.2. Solid phase glycopeptide synthesis . . . . . . . . . . . . . . . . . . . . . 94
2.2.
Synthesis of 0-glycopeptides containing a P-D-xylopyranosyl linkage . . . . . . 101
2.4. Synthesis of 0-glycopeptides with other saccharide units . . . . . . . . . . . . . . . 103
3 . Synthesis of N-glycopeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
110
3.1.
Synthesis of N-glycopeptides containing 2-acetamido-2-deoxy-D-glucose

P-linked to asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
110
3.2
Synthesis of N-glycopeptides containing other sugar residues . . . . . . . . . . 115
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
116
117
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 5. Biosynthesis . I . Introduction
Harry Schnchter . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .

123

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

124


XVI
Chapter 5 . Biosynthesis. 2a. The coenzymic role of phosphodolichols
Frank W. Hemming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Dolichols and other polyprenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Phosphodolichol pathway of N-glycosylation . . . . . . . . . . . . . . . . . . . . . . .
Phosphodolichol structure and biosynthesis: influence on function . . . . . . . . . . . .
Phosphodolichol pathway: subcellular location . . . . . . . . . . . . . . . . . . . . . .
Phosphodolichol pathway: topography in the membrane of rough endoplasmic
reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6 . Mannosyl phosphodolichol: relationships to other glycosylation pathways . . . . . . . .

7 . N-acetylglucosaminyl phosphate transferase . . . . . . . . . . . . . . . . . . . . . . . .
7.1.
Allosteric control by mannosyl phosphodolichol . . . . . . . . . . . . . . . . .
7.2.
Structure-function relationships . . . . . . . . . . . . . . . . . . . . . . . . . .
8. Potential dolichol recognition sequences (PDRSs) of proteins of the phosphodolichol
pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9 . Oligosaccharide and oligosaccharide phosphate produced by the phosphodolichol
pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.
2.
3.
4.
5.

i27

127
127
128
131
131
133
135
135
136
137

139
140
141

Chapter 5. Biosynthesis . 26 . From Glc3Man9GlcNAc2-proteinto MangGlcNAc2-protein: transfer
‘en bloc’ and processing
145
Andre‘ Verbert . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

The transfer reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1
The donor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.
The acceptor site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I .3.
The oligosaccharyltransferase . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 . The deglucosylation steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 . Rough ER a-mannosidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . Golgi rnannosidase I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.

145
145
146
147
148
150
150

151
152

Chapter 5. Biosynthesis. 2c. Glycosyltransferases involved in the synthesis of N-glycan antennae
Harry Schachter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
153

1.
2.
3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biosynthesis of N-glycan antennae . . . . . . . . . . . . . . . . . . . . . .
Galactosyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
UDP-Gal:GlcNAc-R p-l,4-Ga I-transferase (E.C. 2.4.1.38190) . . .
3.1.1. Cloning of p-1,4-Ga 1-transferasecDNA and genomic DNA
3.1.2. Domain structure of glycosyltransferases . . . . . . . . . .
3.1.3. Alternate transcription initiation sites . . . . . . . . . . . .
3.1.4. Expression of recombinant p- 1,CGaI-transferase . . . . . .

. . . . . . . 153
. . . . . . . 154

. . . . . . . 155
. . . . . . . 155
. . . . . . . 155
. . . . . . . 161
. . . . . . . 161


. . . . . . . 163


XVII

164
3.1.5. Targeting to the Golgi apparatus . . . . . . . . . . . . . . . . . . . . . .
UDP-Gal:Gal(pl4)GlcNAc-R (Gal to Gal) a.l,3.Ga Ltransferase
(E.C. 2.4.1.124/151) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
166
3.2.1, Cloning of a - I ,3-GaI-transferase cDNA and genomic DNA . . . . . . . 166
3.2.2. Expression of recombinant a-l,3-Ga1-transferase . . . . . . . . . . . . . 168
Sialyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
168
CMP-sialic acid:Gal(pl4)GlcNAc-R a.2, 6.sialyltransferase (ST6N)
4.1.
(E.C. 2.4.99.1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
169
4.1.1. Cloning of a-2,6-sialyltransferase cDNA and genomic DNA . . . . . . 169
4.1.2. Expression of recombinant a.2, 6.sialyltransferase . . . . . . . . . . . . 171
4.1.3. Targeting to the Golgi apparatus . . . . . . . . . . . . . . . . . . . . . .
172
4.1.4. The sialylmotif . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
172
4.1.5. Chicken CMP-sialic acid:Gal(pl-4)GlcNAc-R a.2, 6.sialyltransferase . 173
CMP-sialic acid:Gal(pl-3/4)GlcNAc-R a.2, 3.sialyltransferases I and I1
4.2.
(ST3N I, E.C. 2.4.99.6, and ST3N 11) . . . . . . . . . . . . . . . . . . . . . . .
173
4.2.1. Cloning of a.2, 3.sialyltransferase I cDNA . . . . . . . . . . . . . . . . . 173

4.2.2. Expression of recombinant a.2, 3.sialyltransferase I . . . . . . . . . . . 173
4.2.3. CMP-sialic acid:Gal( pI-3/4)GlcNAc-R a.2, 3.sialyltransferase I1
(ST3NII) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
174
Brain specific sialyltransferase STX . . . . . . . . . . . . . . . . . . . . . . . .
174
4.3.
CMP-sialic acid:Gal(pl-4)GlcNAc-R a.2, 3.sialyltransferase (STZSAT-3) . . . 175
4.4.
N- Acetylglucosaminyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
175
5.1. UDP-GlcNAc:Man(al-3)R [GlcNAc to Man(al-3)] p.l,2.GlcNA c.
transferase I (EC 2.4.1.101) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
175
5.1.1. Cloning of p-I ,2.GlcNA c.transferase I cDNA and genomic DNA . . . . 175
5.1.2. Expression of recombinant p-I ,2.GlcNA c.transferase I . . . . . . . . . 177
5.1.3. Targeting to the Golgi apparatus . . . . . . . . . . . . . . . . . . . . . .
178
5.1.4. The effect of homozygous ‘knock-out’ of the GlcNAc-transferase 1
gene in transgenic mouse embryos . . . . . . . . . . . . . . . . . . . . .
178
5.1.5. Substrate requirements for p-I ,2.GlcNA c.transferase I . . . . . . . . . . 179
UDP-GlcNAc:Man(a1-6)R [GlcNAc to M a n ( a l 4 ) l p-I ,2.GlcNA c.
5.2.
transferase I1 (E.C. 2.4.1.143) . . . . . . . . . . . . . . . . . . . . . . . . . . .
181
5.2.1. Cloning of p-I ,2.GlcNA c.transferase I1 cDNA and genomic DNA . . . 181
5.2.2. Substrate requirements for p.l,2.GlcNA c.transferase I1 . . . . . . . . . 181
UDP-G~CNAC:R~-M~~(~~-~)[G~CNAC(~~-~)M~~(~~-~)]M
5.3.

[GlcNAc to Man(Pl4)l p.1,4.GlcNA c.transferase 111 (E.C. 2.4.1.144) . . . . . 183
5.3.1. Cloning of p-I .4.GlcNA c.transferase 111 cDNA . . . . . . . . . . . . . 184
5.3.2. Substrate requirements for p.1,4.GlcNA c.transferase 111 . . . . . . . . . 184
5.4. UDP-GlcNAc:R1Man(al-3)R2 [GlcNAc to Man(a1-3)] p-I ,4.GlcNA c.
185
transferase IV (E.C. 2.4.1.145) . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5. UDP-GlcNAc:Ri Man(a1-6)Rz [GlcNAc to M a n ( a l 4 ) l P6-GlcNAc186
transferase V (E.C.2.4.1.155) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
186
5.5.1. Properties of GlcNAc-transferase V . . . . . . . . . . . . . . . . . . . . .
5.5.2. Purification of GlcNAc-transferase V . . . . . . . . . . . . . . . . . . . 187
5.5.3. Cloning of GlcNAc-transferase V cDNA . . . . . . . . . . . . . . . . . 187
3.2.

4.

5.


XVIIl

5.6.

UDP-GICNAC:RI(R~)M~~(~I-~)R~
[GlcNAc to Man(al-6)] P.l,4.GlcNA c.
transferase VI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
188
5.7.
UDP-GlcNAc:GlcNAc(Pl-2)Mana-R[GlcNAc to Mana-] 0- 1,4.GlcNA c.
transferase VI' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

188
6 . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
189
6.I .
The domain structure of glycosyltransferases . . . . . . . . . . . . . . . . . . . 189
6.2.
Localization of glycosyltransferases to the Golgi apparatus . . . . . . . . . . . . 190
6.3.
Genomic organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
191
6.4. Temporal and spatial control of glycosyltransferase activity . . . . . . . . . . . . . 192
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
192
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
193
Chapter 5. Biosynthesis . 3. Biosynthesis of 0-glycans of the N-acetylgalactosamine-a-Ser/rhr
linkage type
201
Inka Brockhausen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.

2.

3.

4.

6.

I.

8.
9.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
201
Structures and distributions of 0-Glycans (GalNAca-Ser/Thr-linked-oligosaccharides). 202
202
2. I .
0-glycan core structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
205
2.2.
Elongated 0-glycan structures . . . . . . . . . . . . . . . . . . . . . . . . . . .
205
2.3.
Terminal structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
205
Functions of 0-glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
206
3. I . Biological roles of rnucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
207
3.2.
Cell adhesion and mammalian lectins . . . . . . . . . . . . . . . . . . . . . . .
208
3.3.
Haematopoietic system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
208
3.4.
Fertilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.
Receptor functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

lntracellular localization and transport of glycosyltransferases and their substrates . . . . 209
212
4.1 .
Initiation of 0-glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . .
215
4.2. Elongation of 0-glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
216
Factors controlling 0-glycan biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . .
218
5.I .
Genetic control of glycosyltransferases . . . . . . . . . . . . . . . . . . . . . .
218
5.2.
Regulation of glycosyltransferase activities . . . . . . . . . . . . . . . . . . . .
219
5.3.
Glycosyltransferase substrate specificity . . . . . . . . . . . . . . . . . . . . . .
220
0-glycosylation: polypeptide a-GalNAc-transferase . . . . . . . . . . . . . . . . . . . .
Synthesis of 0-glycan core 1: core 1 P3-Gal-transferase . . . . . . . . . . . . . . . . . . 222
Synthesis of 0-glycan core 3: core 3 P3-ClcNAc-transferase . . . . . . . . . . . . . . . 224
Synthesis of GlcNAc(CjI-6)Galp- and GlcNAc(@l-6)GalNAc linkages by
p- 1.6-GlcNAc-transferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Synthesis of 0-glycan core 2, 4 and the I antigen . . . . . . . . . . . . . . . . . 226
9. I .
9.1.1. Core 2 P6-GlcNAc-transferases L and M . . . . . . . . . . . . . . . . . 226
9.1.2. Core 4 P6-GlcNAc-transferase activity . . . . . . . . . . . . . . . . . . 230
9.1.3. Blood group I Cj6-GlcNAc-transferase . . . . . . . . . . . . . . . . . . . 231
P- 1,6-GlcNAc-transferases synthesizing linear GlcNAc(P1-6)Gal and
9.2.

232
GlcNAc(P1-6)GalNAc structures . . . . . . . . . . . . . . . . . . . . . . . . .


XIX
10. Synthesis of 0-glycan cores 5 and 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . .
233
Elongation of 0-glycan cores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
234
234
1 1 . I . Elongation P3-GlcNAc-transferase . . . . . . . . . . . . . . . . . . . . . . . . .
Blood
group
i
03-GlcNAc-transferase
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
235
1 1.2.
236
1 1.3. P-1,4-Galactosy I-transferase . . . . . . . . . . . . . . . . . . . . . . . . . . . .
236
1 1.4. p- 1,3-GalactosyI-transferases . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12. Terminal glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
237
238
12.1, cr6-Sialyl-transferase I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
239
12.2. a6-Sialyl-transferase I1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
240
12.3. 0-glycan a3-Sialyl-transferase . . . . . . . . . . . . . . . . . . . . . . . . . . .
243
12.4. Fucosylation of 0-glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
243
12.4.1, a-I,2-Fu c-transferase . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.4.2. a-l,3-Fuc-transferases . . . . . . . . . . . . . . . . . . . . . . . . . . .
244
12.5. Blood group A-dependent a3-GalNAc-transferase and blood group B-dependent
246

a3-Gal-transferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.6. Blood group Cad or Sd-dependent p-1,4-GalNA c-transferases . . . . . . . . . . 247
13. Sulfation of 0-glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
249
249
14. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
250
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
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.
.
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.
.
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.
.
.
.
.
.
.
.
.
.
.
.
References
250
11.

Chupter 5. BioJynthesis . 4u . Gene regulation of terminal glycosylation
Mary Cutlierim Glick . . . . . . . . . . . . . . . . . . . . . . . . . .


261

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
261
Normal regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
261
262
2.1
Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Tissue specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
264
264
2.3.
Chromosomal location . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 . Pathological regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
265
3.1.
Oncogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
265
3.2. Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
267
3.3. Inflammatory response . . . . . . . . . . . . . . . . . . . . . . . . . . .
267
267
3.4.
Genetic diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
268
4 . Molecular mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
268

4.1.
Exons and regulatory elements . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Domain structure and DNA sequence homology . . . . . . . . . . . . . . . . . 272
4.3.
Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
274
274
4.4.
Expression of cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 . Direct relationship of molecular mechanisms to structure/function . . . . . . . . . . . . 275
6 . Additional controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
275
276
7 . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
277
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
277
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
277
I.
2.


xx
Chapter 5. Biosynthesis. 4b. Substrate level controls for N-glycan assembly
Harry Schuchter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

281


The endomembrane assembly line and substrate level controls . . . . . . . . . . . . . .
The roads to highly branched complex N-glycans . . . . . . . . . . . . . . . . . . . . .
Which fork in the road to follow? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Competition for a common substrate . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
GO and NO GO residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. I . The key role of UDP-GlcNAc:Man(a1-3)R [GlcNAc to Man(al-3)]
p-l,2-GlcNA c-transferase I as a GO signal . . . . . . . . . . . . . . . .
3.2.2. NO GO residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
The role of polypeptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

283
283
284
284
285
285

Chapter 5. Biosynthesis . 4c. Compartmentation of glycoprotein biosynthesis
Jiirgen Roth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

287


1.

2.
3.

281
282
282
282
283

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Architecture of cellular organelles involved in glycosylation . . . . . . . . . . . . . .
2.1.
The endoplasmic reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
The intermediate compartment . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
The Golgi apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Techniques to study glycosylation in cells . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Subcellular fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
In situ labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1. Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2. Immunolabeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . Topography of biosynthesis of asparagine-linked oligosaccharides . . . . . . . . . . .
4.1.
General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reactions in the cytosol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2.
4.3.
Reactions in the endoplasmic reticulum . . . . . . . . . . . . . . . . . . . . . .
Reactions in the Golgi apparatus . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.
5. Topography of biosynthesis of threonine-lserine-linkedoligosaccharides . . . . . . . .
5.1. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Site of initial glycosylation reaction . . . . . . . . . . . . . . . . . . . . . . . .
Site of elongation reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

289
290
290
291
291
292
292
292
. 292
292
293
293
300
. 304
304

305
306
306
307
307

Chapter 5. Biosynthesis . 5. Molecular basis of antigenic specificity in the ABO. Hand Lewis
blood-group systems
Winifred M. Watkins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

313

I.

313

1.
2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

287

. 287


XXI
Serology. inheritance and chromosomal location of blood-group genes . . . . . . . . . .
2.1.
TheABOsystem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

The H system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
2.3.
The Sese (Secretor) system . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Lewis system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.
The Ii system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5.
3 . Chemical nature of the blood-group-determinant structures . . . . . . . . . . . . . . . .
3.1.
Early approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Isolation and characterisation of minimal determinant structures . . . . . . . . .
Peripheral disaccharide core structures of A, B, H. Lea,Leb,Le" and L e y
3.3.
determinants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.1. Type 1 and Type 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.2. Type3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.3. Type4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.4. Type6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 4.
Molecules carrying blood-group-determinants . . . . . . . . . . . . . . . . . . .
3.4.1. ABH structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.2. Iistructures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.3. Lea .Leb,sialyl-Lea, Le", L e y and sialyl-Le" structures . . . . . . . . . . .
4 . ChemicalsynthesisofABH. LewisandIi bloodgroupdeterminants . . . . . . . . . . .
5 . Conformation of ABH and Lewis determinants . . . . . . . . . . . . . . . . . . . . . .
6 . Lectin and antibody binding to ABH and Lewis determinant structures . . . . . . . . . .
7 . ABH and Lewis blood group antigen expression in embryonic development, cell
maturation and adult tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8. Biosynthesis of ABH and Lewis determinants . . . . . . . . . . . . . . . . . . . . . . .
9 . The H-gene encoded a- 1,2-L-fucosyltransferase . . . . . . . . . . . . . . . . . . . . . .
Specificity and purificaiton of a-l,2-L-fucosyltransferase in human serum . . . .
9.1.
Molecular cloning of the H-gene encloded a-l,2-L-fucosyltransferase . . . . . .
9.2.
H deficient phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3.
10. The Se-gene encoded a-1,2-L-fucosyltransferase . . . . . . . . . . . . . . . . . . . . .
I I . The A-and B-gene enclosed glycosyltransferases . . . . . . . . . . . . . . . . . . . . .
11 .I. Tissue distribution of A and B transferases . . . . . . . . . . . . . . . . . . . .
11.2. Specificity, cation requirements and pH optima . . . . . . . . . . . . . . . . . .
1 1.3. lsoelectric points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.4. Enzymic basis of Al-Az differences . . . . . . . . . . . . . . . . . . . . . . . .
I I .5. Overlapping specificities of A and B transferases . . . . . . . . . . . . . . . . .
11.6. Antibodies to the A- and B-transferases . . . . . . . . . . . . . . . . . . . . . .
11.7. Purification of A- and B-transferases . . . . . . . . . . . . . . . . . . . . . . . .
1 1.8. Molecular cloning of the ABO locus . . . . . . . . . . . . . . . . . . . . . . . .
1 1.9. Molecular genetic analysis of subgroups and rare variants of A and B blood
groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 1.10. B-like antigen on animal cells and the relationship of bovine and mouse
a- 1,3-galactosyltransferase to human A and B transferases . . . . . . . . . . . .
11.11. Animal genes cross hybridising with human ABO genes . . . . . . . . . . . . .
12. The Le-gene encoded a- 1,3/1/4-fucosyltransferase . . . . . . . . . . . . . . . . . . . .
2.

314
314
315
316

317
319
320
320
321
323
323
323
324
324
325
325
329
330
331
331
332
334
336
339
339
340
341
344
347
347
347
348
348
349

350
351
352
355
357
358
359


XXII

12.1. Purification and properties of the a.l,3/1, 4.fucosyltransferase . . . . . . . . .
12.2. Molecular cloning of the Le-gene encoded transferase (Fuc-TIII) . . . . . . .
12.3. Molecular genetics of the Lewis-negative phenotype . . . . . . . . . . . . . .
1 3. a-1,3-Fucosyltransferases (Fuc-TIV, Fuc-TV,and Fuc-TVI) . . . . . . . . . . . . . .
13.1. Purification and properties of a-1,3-fucosyltransferases . . . . . . . . . . . . .
13.2. Relationship between the Lewis-gene associated a-1,3/1,4-fucosyltransferase
and the a-I , 3-fucosyltransferase in plasma. . . . . . . . . . . . . . . . . . . .
13.3. Molecular cloning of a-I , 3-fucosyltransferase genes (Fuc-TIV, Fuc-TV and
FUC-TVI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14. Biosynthesis of i/I structures and cloning of the branching p-l,6-N-acetylglucosaminyI.
transferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15. Blood group antigens and disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16. Function of ABO and Lewis blood group structures . . . . . . . . . . . . . . . . . . .
17. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Addendum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chapter 5. Biosynthesis. 6. The role of polypeptide in the biosynthesis of protein-linked
oligosaccharides

T. Camphausen. Hsiang-ai Yu and Dale A . Cumming . . . . . . . . . . . . .
Kcz~~ritoncl

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.
Historical perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2. Terms and concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3
Other participating factors . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 . Evidence that the polypeptide chain specifies attached oligosaccharide structure . . . .
2.1
Protein structural features . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Comparison of identical glycosylation sites for proteins expressed in
heterologous systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Glycosylation and protein folding . . . . . . . . . . . . . . . . . . . . . . . .
2.4.
Molecular manipulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 . Mechanistic models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1,
Steric accessibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Kinetic efficiency model . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Site-directed processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . A unifying synthesis: ‘site-specific’ topological modulation . . . . . . . . . . . . . . .
5 . Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.


359
362
363
364
364
366
367
368
369
371
373
374
375
376

391
391
392
392
393
394
394
395
397
398

398
398
400

403
406
407
412

Chapter 5. Biosynthesis. 7. How can N-linked glycosylation and processing inhibitors he used to
study carbohydrate synthesis andfinction
Y.T. Pan and Alan D . Elbein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
415
1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

415


XXIII
Inhibitors of lipid-linked saccharide formation . . . . . . . . . . . . . . . . . . . . . . .
419
2.1.
Tunicamycin and related antibiotics . . . . . . . . . . . . . . . . . . . . . . . .
Other antibiotics that inhibit lipid-linked saccharide formation . . . . . . . . . . 423
2.2.
Sugar analogues and amino sugars . . . . . . . . . . . . . . . . . . . . . . . . .
426
2.3.
428
2.4.
Glucose starvation and energy charge . . . . . . . . . . . . . . . . . . . . . . .
3 . Inhibitors of processing glycosidases . . . . . . . . . . . . . . . . . . . . . . . . . . . .

428
3.1,
Inhibitors of glucosidase I and glucosidase I1 . . . . . . . . . . . . . . . . . . . 430
3.1.1. Castanospermine, Deoxynojirimycin and DMDP . . . . . . . . . . . . . 430
435
3.1.2. Australine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
435
3.1.3. Alexine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.4. DIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
435
3.1.5. MDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
435
435
3.1.6. Bromoconduritol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Inhibitors of Mannosidase I and Mannosidase I1 . . . . . . . . . . . . . . . . . . 436
3.2.1. Deoxymannojirimycin . . . . . . . . . . . . . . . . . . . . . . . . . . .
436
436
3.2.2. Kifunensine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.3. D-Mannonolactarn amidrazone . . . . . . . . . . . . . . . . . . . . . .
438
3.2.4. DIM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
438
3.2.5. MMNT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
438
3.2.6. Mannostatin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
438
3.2.7. Swainsonine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
439

Inhibitors of dolichyl-P synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.
440
441
4.1.
25-Hydroxycholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Compactin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
441
5 . Inhibition of protein targeting or movement . . . . . . . . . . . . . . . . . . . . . . . .
442
5.1.
Monensin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
442
5.2.
BrefeldinA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
443
6 . Compounds that modify protein structure or synthesis . . . . . . . . . . . . . . . . . . . 443
6.1.
P-Hydroxynorvaline and fluoroasparagine . . . . . . . . . . . . . . . . . . . . .
443
444
6.2.
Inhibitors of protein synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . .
445
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
445
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.


Chapter 6. Bacterial glycoproteins
Manfred Sumper and Felix T. Wieland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.

2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Glycoproteins as constituents of bacterial S-layers . . . . . . . . . . . . . . . . . . . . .
2.1.
Archaebacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.1, Halobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1. Methanogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Eubacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Glycoproteins as constituents of flagellins . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Halobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Methanogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

455
455
455
456
456
460
460
461

461
462


XXIV
Glycosylated exoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
462
Biosynthetic aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
462
Biosynthesis of the cell surface glycoprotein and the flagella of halobacteria . . . 463
5.1.
5.1.1. Sulfated glucosaminoglycan . . . . . . . . . . . . . . . . . . . . . . . .
463
5.1.2. Sulfated oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . .
464
5.1.3. Model for the biosynthesis of N-linked saccharides in halobacteria . . . 465
5.2.
Methanothermus fervidus glycoprotein . . . . . . . . . . . . . . . . . . . . . .
467
5.3.
S-layer glycoprotein of Bacillus alvei . . . . . . . . . . . . . . . . . . . . . . .
468
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
469
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
470
470
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.
5.


Chapter 7. Protein glycosylation in yeast
L. Lehle and W. Tanner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.
2.
3.
4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Yeast mannoproteins and cell wall architecture . . . . . . . . . . . . . . . . . . . . . .
N-linked carbohydrate structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
N-glycosylation pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Lipid-linked oligosaccharide assembly . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Core glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
N-glycosylation sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Maturation to inner core and outer chain biosynthesis . . . . . . . . . . . . . .
4.4.
4.4.1. Inner core modificaton . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.2. Outer chain attachment . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 . The 0-glycosylation pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
Biosynthesis, molecular biology . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Defined 0-mannosylated proteins . . . . . . . . . . . . . . . . . . . . . . . . .
6. Protein modification by GPI-anchors . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7 . Topology and compartmental organization of glycosylation reactions and the
secretory pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8.
Specific topics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.1.
Glycoproteins and cell/cell recognition . . . . . . . . . . . . . . . . . . . . . .
8.2.
Cell wall glycoproteins and killer toxin action . . . . . . . . . . . . . . . . . .
8.3.
Relevance of N- and 0-glycosylation in general . . . . . . . . . . . . . . . . .
9. Protein glycosylation in other fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10. Heterologous expression and glycosylation of proteins . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

475
475
476
479
480
480
481
482
. 483
483
484
486
486
489
490
491

494
494
. 495
. 495
496
. 491
500
501
501

Chapter 8. 0-glycosylation in plants
Frans M . Klis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

511

1.

511

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .