.___‘ellular and Molecular Life
`c
`I" I
`
`NATURAL SCl‘ENCES
`
`mantras:
`
`:u'rwrsr‘m'i
`
`APR 29‘ 1999
`
`In thi
`
`Lin-mes
`
`Reviews
`
`lmmunotolerant functions of HLA-G
`E. D. Carosella, J. Dausset and N. Rouas-Freiss
`
`Antisense RNA gene therapy for
`studying and modulating biological
`processes
`B. Weiss. G. Davidkova and L-W. Zhou
`
`Structural view of cadherin-mediat
`cell-cell adhesion
`J. R. Alattia. H. Kurokawa and M. lkura
`
`Eukaryotic glycosylation: whim of
`nature or multipurpose tool?
`G. Reuter and H.—J. Gabius
`
`Peptidyl—prolyl cis—trans isomerases
`superfamily of ubiquitous folding
`catalysts
`S. F. Giithei and M. A. Marahiel
`
`Roles of the DNA mismatch repair .
`nucleotide excision repair proteins
`
`during meiosis
`D. T. Kirkpatrick
`
`
`
`- Vol. 55
`' Pp. 327-511
`I]
`'El 1999
`l420-582X
`
`Page 1 of 59
`
`CSL EXHIBIT 1101
`
`Page 1 of 59
`
`CSL EXHIBIT 1101
`
`

`

`Ellular and Molecular Lift: Science
`
`as."
`
`
`
`
` formerly Experientia
`
`Scope
`
`The multidisciplinaryjournal CflrfLS, Cellular andMolecular Life Sciences (formerly Experientia) publishes
`articles, mini-reviews, reviews and multi-author reviews covering the latest aspects of biological and biomedi
`research. The journal will consider contributions focusing on molecular and cellular aspects of biomedicine,
`biology, immunology, molecular genetics, neuroscience, biochemistry, pharmacology, and physiology related
`pharmacology.
`
`'
`
`
`
`Editor-in-chief
`
`Editorial Board
`
`Advisory Board
`
`C.C.F. Blake, Oxford
`D. Arigoni, Zfirich
`E. Carafoli, Zfirich
`TA. Biclrle, Basel
`KJA. Davies, Los Angeles, CA J. Engel, Basel
`G. Dirheimer, Strasbourg
`U. Feige, Thousand Oaks,
`T. Imoto, Fukuoka
`J.-M. Frere, Liege
`H. Jfimvall, Stockholm
`M. Go, Nagoya
`E. Kubll, Zfirich
`T. Kirchhausen, Boston, MA
`H.-G. Rammensee, Tiibingen
`K. Kuchler, Wien
`R. Timpl, Marflnsried
`F. Mayor In, Madrid
`W. Wabli, Lausanne
`R.I. Norman, Leicester
`M. Parnham, Zagreb
`P. Potier, Gif-sur-Yvette
`I. Slaninovfi, Praha
`P. Venetianer, Szeged
`
`Professor Dr. P. Jolles
`Laboratoire de Chimie
`des Substances Naturalles
`URA CNRS No. 401
`Museum National d’Histoire Naturelle
`63, rue Buffon
`F-75005 Paris I France
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`Cover
`
`
`
`llhistration of a low-energy conformation ofN-aoetyllactosamine (lacNAc), a common terminal section of N—glycans. It
`me.g.bebmmdbymmflimlwfim(galmfins),safingmdmldngmmt(hnmymowdedbym. C.-W.vonderLieth.
`Heidelberg, Germany).
`G. Rutter and H.-J. Gabius, p. 368, this issue.
`
`—————_—_____________________
`
`Page 2 of 59
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`Page 2 of 59
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`

`

`- SSINO. 3, 1999
`327#511
`
`
`.March 1999
`
`-:_
`
`
`
`
`Immunotolerant functions of HLA-G
`E. D. Caroseita, J. Dausset and N. Rouas-Freiss
`(France)
`
`Antisense RNA gene therapy for studying and
`modulating biological processes
`B. Weiss. G. Dauidkova and L.-W. Zhau
`(USA)
`
`Structural
`adhesion
`
`view of cadherin-mediated cell-cell
`
`J. R. Aiattia, H. Kurakawa and M. ikura
`(Canada)
`
`Eukaryotic glycosylation: whim of nature or
`multipurpose tool?
`G. Renter and H.-J. Gabius
`(Germany)
`
`Peptidyl-prolyl sis-trans isomerases, a superfamily
`of ubiquitous folding catalysts
`.S'. "F. Garnet and M. A. Marahiet
`(Germany)
`
`Roles of the DNA mismatch repair and nucleotide
`excision repair proteins during meiosis
`D. T. Kirkpatrick
`USA
`(
`)
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`' arch Articles
`
`456
`
`Phorbol ester exposure activates an ARI-mediated
`increase in ERGO-l messenger RNA expression
`in human ovarian tumor cells
`Q. Li, L. Zhang, B. Tsang, K. Gardner,
`F. Bostick-Bruton and E. Reed
`(USA)
`
`467
`
`Protein kinase-dependent cverexpression of the
`nuclear protein pirin in c-J UN and RAS
`transformed fibroblasts
`
`A.-C. Bergman, A. A. Aiaiya, W. Wendter,
`B. Binetruy, M. Shoshan, K. Sakagucht',
`T. Bergman, U. Kronenwett, G. Auer.
`E. Appetta, H. Jiirnuatt and S. Linder
`(Sweden, Germany, France and USA)
`
`Repetitive use of a phosphate-binding module in
`DNA polymerase ,6. Oct-l POU domain and
`phage repressors
`K. Yum, M. Shianyu. K. Kawatani and M. Gt?
`(Japan)
`
`Alloxan acts as a promidant only under reducing
`conditions: influence of melatonin
`H.-J. Brb‘mme, H. Ebeit, D. Pesehice and
`E. Pesehke
`
`(Germany)
`
`Repolarization abnormalities in cardiomyocytes of
`dogs with chronic heart failure: role of sustain-
`ed inward current
`A. I. Undrcvinas, V. A. Maitsev and
`H. N. Sabban
`(USA)
`
`The involvement of the renin-angiotensin system in
`the regulation of cell proliferation in the rat
`endometrium
`M. Pawlikowski, G. Maren-Murat! and
`S. Mucna
`
`472
`
`487
`
`494
`
`506
`
`
`
`A common
`
`ancestor
`
`for
`
`a
`
`subunit
`
`in
`
`the
`
`(Potand)
`
`mitochondrial proton-translocating NADH:
`ubiquinone oxidoreductase (complex I) and
`short-chain dehydrogenasesireductases
`M. E. Baker, W. N. Grundy and C. P. Eikan
`(USA)
`
`511
`
`Announcement
`
`The Instructions for Authors appear on the inside back
`cover of this issue
`
`Page 3 of 59
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`Page 3 of 59
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`
`
`Page 4 of 59
`
`

`

`
` CMLS Cellular and Molecular Life Scion '
`
`CMLS, Cell. Mol. Life Sci. 55 (1999) 368-422
`1420-682XI99f030368-55 $ 1.50 + 0.20.“)
`© Birkhauser Verlag. Basel. 1999
`
`Review
`
`
`rather common type of posttranslational
`
`Eukaryotic glycosylation: whim of nature or multipurpose
`tool?!"
`
`G. Reutert and l-l.-J. Cabins“
`
`lnstitut fiir Physiologische Chemie, Tierarztliche Fakultat, Ludwig-Maximilians-Universitat, Veterinarstr. 13,
`D-80539 Miinchen (Germany), Fax +49 89 21802508, e-mail: gabius@tiph.vetmed.uni-muenchen.de
`
`Received 2 October 1998; received after revision 1? November 1998: accepted 1? November 1998
`
`Abstract. Protein and lipid glycosylation is a ubiquitous
`phenomenon. The task of cataloguing the great struc-
`tural variety of the glycan part has demanded consider-
`able efforts over decades. This patient endeavor was
`imperative to discern the inherent rules of glycosylation
`which cannot affirm assumptions on a purely coinciden-
`tal nature of this type of protein and lipid modification.
`These results together with theoretical considerations
`uncover a salient property of oligosaccharides. In com-
`parison with amino acids and nucleotides, monosaccha—
`rides excel in their potential to serve as units of hardware
`for storing biological information. Thus, the view that
`glycan chains exclusively affect physicochemical proper—
`ties of the conjugates is indubitably flawed. This original
`concept has been decisively jolted by the discovery of
`endogenous receptors (lectins) for distinct glycan epi-
`topes which are as characteristic as a fingerprint or a
`
`signature for a certain protein (class) or cell type. Recent
`evidence documents that these binding proteins are even'
`endowed with the capacity to select distinct low-energy
`conformers of the often rather flexible oligosaccharides,
`granting entry to a new level of regulation of ligand
`affinity by shifting conformer equilibria. The assessment
`of the details of this recognition by X-ray crystallogra-
`phy, nuclear magnetic resonance spectroscopy. mi-
`crocalorimetry and custom-made derivatives is supposed
`to justify a guarded optimism in satisfying the need for
`innovative drug design in antiadhesion therapy.
`for
`example against viral or bacterial
`infections and un-
`
`wanted inflammation. This review presents a survey of
`the structural aspects of glycosylation and of evidence to
`poignantly endorse the notion that carrier~attached gly-
`can chains can partake in biological information transfer
`at the level of cell compartments. cells and organs.
`
`Key words. Cell adhesion; drug design; glycan metabolism: glycolipids; glycoproteins; glycosaminoglycans; lectins;
`recognition.
`
`Introduction
`
`Proteins are ubiquitous compounds in all living organ-
`isms with many well-established biological functions.
`They play important roles, for example as biocatalysts,
`transport and targeting devices, in establishing cell-type
`
`
`* Dedicated to Prof. Dr. F. Cramer on the occasion of his 75th
`birthday.
`** Corresponding author.
`1‘ Present address: Haferkarnp 28, D-24145 Kiel (Germany).
`
`Page 5 of 59
`
`specificity and in various aspects of cell sociology. Post-
`translational modification of proteins adds a further
`level of regulation to their activity and can thus enlarge
`their capacity to take part in regulatory events. The
`reversibility of the introduction of small
`inorganic
`groups into amino acid side chains such as phosphate
`allows proteins to be used as molecular switches. Evi-
`
`the capacity of the peptide backbone is not
`dently,
`restricted to serving as acceptor for a phosphate group-
`Another
`
`Page 5 of 59
`
`

`

`
`. Cell. Mol. Life Sci. Vol. 55. 1999
`
`
`
`
`3.: aification is protein glycosylation. A potential indi-
`" on of a biochemical role of the carrier-immobilized
`
`units is furnished by the observation that a sec-
`
`abundant class of biomolecules shares the property
`proteins to be decorated with oligosaccharides. Cer-
`
`-'de-based lipids as major constituents of biomem-
`
`es can carry attached glycan chains. and the
`
`:e._phatide phosphatidylinositol with its lipid anchor
`
`'
`and hydrophiiic inositol head group is likewise
`
`ed as a means of glycan attachment. Glyco-
`
`teins and glycolipids are referred to as glycoconju-
`
`-
`and members of
`the group of
`complex
`
`
`_. parts are directed into the outer environment and
`
`constitute the glycocalyx. While the glycan chains
`
`pids rarely extend beyond a dozen monosaccharide
`
`
`
`those of glycoproteins can protrude further
`ace,
`the cell membrane due to either long giycan chains
`'_
`
`_-.linkage to an amino acid at a remote position within
`
`peptide chain.
`
`the early years of research on these compounds,
`
`
`
`for any taking stock of functional aspects. Now a
`
`of sophisticated and further continuously improved
`
`iods is available, consequently making it easier to
`
`_
`the initially puzzling complexity of saccharide se-
`
`_‘i.-ces on record [145]. Only after having pursued
`
`- _ctural analysis, which revealed a wide range of
`
`ii can structures. could attention he turned to the enzy—
`'
`routes of biosynthesis and to the delineation of a
`
`-
`ive physiological meaning, these lines of reasoning
`
`acted, for example, in reviews by Roseman [7] and
`
`lnterburn and Phelbs [8]. This historical development
`
`the different phases of research on complex carbohy-
`
`tes is'niceiy described by a series of authoritative
`
`_ ews [9— 18]. Currently, the enormous strides taken in
`
`T
`- area of sugar-mediated recognition suggest
`that
`
`’3; can constituents of proteins and lipids establish an
`i'uhabetical system, adding to the well-accepted coding
`
`'g'ai nortance of nucleic acids and peptide backbones [19,
`
`'- Citing from the article of Winterburn and Pheibs
`
`.-;.'-i, carbohydrates are ideal for generating compact
`
`ts with explicit
`informational properties since the
`
`utations on linkages are larger
`than can be
`
`hieved by amino acids, and, uniquely in biological
`
`
`.i-i-tnoe of the glycosyl residues is to impart a discrete
`
`ugnitional role on the protein’ (or any other car‘rier
`ckbone). This notion has meanwhile continuously
`ived enough support to keep it
`in the eye of a
`._
`adership from different areas of life sciences [1, 21—
`
`
`
`
`
`Page 6 of 59
`
`Review Article
`
`369
`
`tures. analysis, conformation and biosynthesis, and out-
`line the current concept of the biological role of these
`fascinating molecules.
`
`The sugar code
`
`The term ‘complex carbohydrates’ should not solely be
`thought to connote a technically demanding task of
`cataloguing structures. Principally, oligomers of any
`class of biomolecules can establish a coding system for
`information storage, resembling words in a language. In
`this sense, structural complexity entails the intriguing
`capacity to serve as versatile hardware for biological
`information transfer. The capacities for this duty are
`not equal for the biochemical coding systems. Amino
`acids and nucleotides are linked nearly exclusively in
`
`H0
`
`0H
`0
`
`0H OH
`
`HO
`
`0H
`0 OH
`
`OH
`
`OH
`
`a-iuranosc \ / B turanoee
`”\c/xo
`H-c-OH
`ao-c-a
`H-
`-OH
`ӣ3-01.
`cazoa
`
`5:
`
`ct - pyranose
`
`HO
`
`0
`
`HO
`
`open chain
`(Fischer projection]
`
`£3
`
`[5 - pyranose
`
`[10
`
`0mo"H0
`
`Reeves confounation
`4'31
`
`Figure l. Mutarotation of D-glucose with the different representa-
`tions of D-glucose as Fischer projection. Haworth projection and
`Reeves ‘C. chair conformation
`
`Page 6 of 59
`
`

`

`370
`
`G. Renter and H.—J. Gabius
`
`linear chains via peptide or phosphodiester bonds.
`Only the primary sequence of the monomeric building
`units in the chains allows for variability in these two
`cases.
`This
`situation
`changes
`drastically when
`cligosaccharides are considered. It
`is pertinent to re-
`call aspects of the basic biochemistry of sugars to ap-
`preciate these extraordinary properties of this type of
`oligomer, which can reach an unprecedented level of
`storage capacity.
`
`of
`teaches mutarotation
`biochemistry
`Textbook
`monosaccharides such as D-glucose primarily from the
`perspective of a measurable physical parameter {opti-
`cal rotation). Since linear forms of. for example. any
`hexose are normally present only in minute amounts,
`hemiacetals or -ketals are invariably found. The mu-
`tarotation of a hexose yields four different ring struc-
`tures for each monomeric unit. as depicted in figure 1
`for D—glucose (Gle). Viewed from the perspective of
`evaluation of hardware abilities, this result is of rele-
`vance. Similarly far~reaching is
`the fact
`that each
`monosaccharide presents a variety of hydroxyl groups.
`Formation of a bond between two monosaccharide
`units by condensation can engage diverse possibilities,
`a daunting task for the organic chemist. When the
`anomeric center of a monosaccharide participates in
`the glycosidic bond, each anomer can theoretically re-
`act with one of the five, chemically nearly equivalent
`hydroxyl groups of another hexopyranose unit. Figure
`2 shows that permutations on the basis of only these
`
`
`
`Figure 2. Possible linkage points for a disaccharide consisting of
`Ga] and Ole. The different structures that can be formed result
`from variations in (i) anomeric configuration (a: or 5}. (ii) ring
`size. that is {uranose or pyranose, with only pyranose depicted in
`the figure {see fig.
`I).
`(iii)
`linkage points as indicated by the
`arrows. with the dotted line for the formation of nonreducing
`disaccharides involving both anomeric centers, and {iv} the se-
`quence of the two monomers. Excluding linkage via the two
`anomeric centers. each C! hydroxyl group can be conjugated to
`any of the four available hydroxyl groups at C2, C3. C4 and C6
`in a- or fi-configuration. yielding 8 isomers for each of the two
`anomeric centers.
`
`Page 7 of 59
`
`
`
`in which the two anomeric centers are linked to -
`
`other. Sequence permutation doubles this number,
`5.;
`this calculation. the potential for alteration of the '
`size (pyranoselfuranose, see fig. 1) has not even beefi,
`
`incorporated. Using two amino acids or nucleoti -...;
`only two different structures can be generated.
`
`If we allow the formation of any linear or bran l.'-'|__il
`trisaccharide from a pool of 20 different monosacchug
`
`'
`rides, we end up with 9 >< 10" possibilities. whe
`
`only 8000 tripeptides can be formed from the
`
`amino acids [19]. For a hexamer this gap widens to?
`
`more than seven orders of magnitude. This enormous.
`coding capacity of oligosaccharides
`renders
`them-
`highly suitable hardware for
`information storage,
`However,
`it
`is essential
`to add that
`the theoretical
`number of combinations has by far not been un-
`earthed in nature, probably because the equally high-
`number of corresponding glycosyltransferases is not-
`available in the organism [5. 27—29]. A compensatory
`diversification is found instead by addition of ‘small
`substituents’ such as phosphate, acetate or sulfate [19,.
`26. 30m38]. As will be illustrated later
`in severei'
`
`
`
`
`
`places, single or multiple modifications with such sub-
`stituents can even occur on one monosaccharide.
`residue, affecting its character as code letter. For ex;
`ample,
`the 4-sulfation of N-acetyl-D-galactosamine
`(GalNAc) can be likened to the formation of an um—
`
`laut in German language. If now multiple saccharide
`chains, each containing a set of possible structural
`variations, are attached to one protein backbone as is-
`the case for many glycoproteins, one can grasp the
`immediate relevance of these calculations and findings
`for the theoretical content of information stored in the-
`glycoconjugate.
`Posttranslational modification by glycosylaticn can.
`even lead to the creation of discrete subsets of glyco-
`proteins (glyccforms. according to Rademacher et a].
`[39]). They share an identical polypeptide sequence but
`differ in the glycan composition. As will eitemplarilyIr
`be outlined in the section on glycans and disease.
`monitoring the relative proportions of serum transfer-
`rin glycoforms has diagnostic value. It is indicative of
`clinically defined inherited syndromes or of impair-
`ment of liver function by distinct causes. In line with
`the given reasoning,
`it
`is tempting to speculate that
`this potential is not wasted as a costly whim of nature
`but
`rather establishes a multipurpose tool
`that
`is
`taken advantage of in diverse circumstances. Before
`the functional aspect can be discussed. it is essential to
`provide an insight into the structural organization of
`protein and lipid glycosylaticn.
`
`Page 7 of 59
`
`

`

`
`
`k:
`
`
`l.Mol.Life Sci. VOLSS. 1999
`
`Review Article
`
`371
`
`H30
`
`H0
`
`‘.
`
`.'
`
`“*0
`
`0
`
`OH
`
`O
`
`NHAc
`
`NH
`
`’0
`\c/
`
`l
`
`KfNH
`Hts/0'20
`
`0%?
`
`NH
`
`X
`
`o
`
`C-‘c:
`
`o
`
`./
`
`
`NHAc
`
`\NH
`
`
`
`3. Linkage points for the covalent attachment of common
`d Owglycans to protein. The GlcNAcfi l-Asn-X-Ser sequon is
`eristic for most N-glycans. The carbohydrate chain is
`a at the C-4 of GlcNAc as indicated by the wavy line {a};
`.- of core fucosylation, see figure IS. The GalNAcxl-Ser
`re of mucin-type O-linked glycoproteins can be extended
`tion of further saccharides to C-3 and C-6, leading to the
`'_uction of branches (.5).
`
`
`
`
`
`w a attachment to proteins
`
`
`
`-,_glycoproteins. the glycan chains are nearly always
`"a
`= ntly linked to suitable functional groups of amino
`
`in side chains within a protein. The elucidation of the
`_=:"-" urea '01“ the linkage region between carbohydrate
`_ protein was
`initiated with the description of
`
`NAcfil-Asn for N-glycans and GalNAcrxl-Ser or
`..':'_'for O—glycans (fig. 3) [1, 10. 25, 26, 40]. Ensuing
`:J rch considerably extended our knowledge in this
`
`
`
`re 4. Structure of the «4Xylfi l-Ser linkage of glycosaminogly—
`4":
`'_=-I s.
`
`
`
`Page 8 of 59
`
`a
`
`b
`
`H0
`
`H
`
`Ho
`
`H0
`
`H0
`
`HO
`
`3‘
`
`b
`
`6
`
`5
`
`HN/
`Mcéo
`L__
`
`/5
`HN
`°\/kc4’o
`l
`
`OH
`
`0H
`
`0
`
`0H
`
`OH
`
`.
`
`HN/
`Ody/0
`I.
`
`OH
`
`NHAc
`
`0
`
`OH
`
`N
`
`o
`
`H\\c//
`
`l.
`
`NH
`
`:1
`
`/C=:.-o
`
`Figure 5. Less common Ser glycosylations are found in the (a)
`-3Fucal-Ser linkage and (b) Glcfi l-Ser linkage in EGF-Iike do-
`mains. Another possible mode of O-glycosylation as well as one of
`glucose attachment to protein are presented by the linkages (c)
`GlcNAcfil-Ser and (d) Glcfll-Asn. Type (c) is predominantly
`found in intracellular proteins in the nucleus and cytoplasm.
`
`area [15. 28. 29, 41, 42]. The current status of delin-
`eation of the rules that govern the selection of suitable
`glycosylation sites in the peptide sequence has enabled
`explanation of the nonrandom nature of this glycan
`attachment. It has been recognized that for N-glycosidic
`linkage a peptide sequence of Asn-x-Serf'l‘hr (a sequon)
`is required, in which the variable amino acid X seems to
`be important for the efficiency of recognition of the
`resulting sequon [43—45]. Additionally, the amino acid
`following a sequon (the Y-position in Asn-X-Serfl'hr-
`
`Page 8 of 59
`
`

`

`3?2
`
`G. Renter and H.-.l. Gabius
`
`Y) has a notabIe impact on glycosylation efficiency,
`especially for Asn-X-Ser sequons, Pro, Glu and Trp
`causing the most negative effect [46. 47]. For human and
`bovine plasma proteins C. human von Willebrand fac-
`tor and human CD69 an unusual Asn-X—Cys sequon
`has been identified for N~glycan attachment [48, 49].
`The definition of the sequon allows potential glycosyla-
`tion sites in any protein to be deduced on the basis of
`amino acid sequence data. No equivalent peptide se-
`quence requirement has been described so far for classi-
`cal mucin-type O-glycosylation. The initiation of chain
`growth by several transferases with varying degrees of
`stringency of substrate recognition has at least suggested
`general rules and an algorithm of predictive value [50.
`51]. A substantial functional redundancy of these trans-
`ferases was inferred by mutagenic recombination of one
`gene with a deletion in the catalytic region, which did
`not
`impair O~glycan synthesis [52]. The lack of an
`0~glycosylation sequon does not mean that the reaction
`of the uridine diphosphate (UDP)-GalNAc:polypeptide
`N-acetngaIactosaminyltransferases can take place with-
`out sequence requirements. The spatially accessible ac-
`ceptor
`amino
`acid
`appears
`to
`be
`surrounded
`preferentially by Ser, Thr, Pro. Ala and Gly residues
`[28, 29, 50, 53]. Serfl‘hr residues not only serve as
`O-glycan acceptors but also as adaptors for another
`class of glycans. The third type of complex carbohy-
`drates are the glycosaminoglycans using the glycan-
`protein linkage 4Xylfi l-Ser/Thr (fig. 4} [54]. Usually, a
`trisaccharide 4Gchfl l-3Cialfil-3Cialfil- is attached to
`the 4-position of Xyl before the typical building blocks
`of the individual glycosaminoglycan are added [55].
`In addition to allowing GalNAc and Xyl attachment,
`the hydroxyl groups of Serfl‘hr are sites for the incorpo-
`ration of other glycan modifications. Despite their com-
`paratively low frequency they should not be overlooked
`in this context. On the contrary,
`they illustrate the
`existence of cytoplasmic glycosylation pathways inde-
`pendent of these classical routes, each case an economic
`argument against a merely superfluous expression of the
`engaged glycosyltransferases. Epidermal growth factor-
`(EGF)-like domains of several glycoproteins including
`blood coagulation factors VII. IX and X11, plasminogen
`activators and protein Z are the targets of a sequon-de-
`pendent O-fucosylation and O—glucosylation [56. 57].
`The enzymatic introduction of oc-L-fucose (a-L-Fuc);
`(fig. 5a) can initiate extension to GIcfll-3Fucat l-SerfThr
`or to a sialylated N—acetyllactosamine sequence at the
`3-position [58, 59]. The sequon dependence of fucosyla-
`tion in the EGF-Iike domains of tissue-type plasmino-
`gen activator (t-PA) and urokinase was reinforced by
`the absence of this modification in the human chimeric
`plasminogen activator Kztu-PA lacking this sequence
`element [56, 60]. The consensus'sequence for the reac-
`tion of this fuoosyltransferase is Cys-X-X-GIy-Gly—Serf
`
`Eukaryotic glycosylatioq
`
`Thr-Cys-, which can also be found in other types of
`proteins besides EGF module-bearing proteins [56, 591
`Once added, protein-bound L-l‘ucose has been intimated
`to be important for mediating binding of the fucosy.
`lated protein to human HepGZ hepatoma cells, because
`fucose-free EGF-like domains lack this capacity [6]].
`Since binding and degradation of IPA is not inhibited
`by 50 mM L-fucose in the case of human and rat
`hepatoma cells [62],
`this suggestion requires further
`experimental support. Another possibility concerns the
`
` 3.
`
`OH
`
`0
`
`OH
`
`H
`
`H
`
`/
`
`0'
`HN
`(Kg/O\/i\C/I’
`é',
`l
`
`o
`
`Ho
`
`HO
`
`0H
`
`0
`
`H
`
`,
`
`/'
`
`HN
`
`'3
`
`c
`
`Figure 6. C—C linkage of Man and Trp in human RNase {ri-
`bonuclease) U, (a). This is currently the only example known
`without N- or O-glycosidic linkage between saocharide and
`protein. Neither biological implications nor enzymatic ways for
`biosynthesis are known so far. Structure of the Manal-P-Scl‘
`linkage as an example of phosphoglycosylation (b). Instead of
`D-mannose, Fuc, GlcNAc and Xyl may also be found. ManaI-Ser
`linkage as common glycosylation motif in yeast (:1.
`
`
`
`Page 9 of 59
`
`Page 9 of 59
`
`

`

`
`-:-'._.-: --Cell. Mol. Life Sci. Vol. 55. 1999
`
`Review Article
`
`373
`
`R
`l?
`o
`"Ca A xHN
`ii
`I'M
`(Ii wO/$?O/
`°
`
`.
`
`m1—2Mmu1fihm140bflataslmmt
`3
`1
`Gabtecdutmt
`2
`|
`Saint
`
`{Gm-9L6
`0
`o
`l
`E
`"
`TOIETNO/ \(CHIJuCHs
`,(cuzmcrs
`0x.
`5'le
`fi
`o
`
`
`
`ules are embedded in the cell membrane via the fatty acids (often
`of this Family of molec
`7. GPI anchor structure. All members
`JI'acylglyool. Starting with D-glucosamine.
`of the phospholipid part which can comprise ceramide. mono- or diacylglycerol or alkyl
`hed to this membrane-embedded anchor
`asaccharide that varies structurally among the different glycoproteins is covalently attac
`each central Denannose unit is a major source of structural
`phosphoethanolamine} at
`rporation of substituents (saccharides,
`al-Z-linked mannose residue of the core glycan.
`
`.
`ethanolamine is introduced to the
`hydrophobic C-terminus. which generates the
`
`idation follows cleavage of the peptide backbone 10412 residues away from the
`Asn, Gly. Ala. Ser or Cys), yielding its glypiation. Potential
`
`terminus. A protein is attached by its w-site (preferentially Asp.
`f a modification at the inositol residue will hamper Pl-PLC
`-_r sites for (G)PI-PLC and GPl-PLD are indicated. Introduction 0
`
`(G)Pl-PLCID: (glycosyl) phosphatidylinositol phospholipases CID.
`
`
`
`
`'ve influence of fucosylation at ThrlS on the
`h factor activity of the EGF-like domain in hu-
`uroltinase-type plasminogen activator [63. 64]. As
`
`ted above. the EGF module for example in t-PA
`
`_ not only harbor the 0-fucosy1ation. It also carries
`
`'j' O-linked glucose unit (fig. Sb) in the consensus
`
`'1 ence Cys-X-Ser-X~Pro-Cys of the EGF domain [56.
`
`.1". The glucose moiety may further be substituted at
`
`'
`' -position by Xylrxl-3Xylotl-, as found in blood
`
`tion factors VII and IX, protein 2, t-PA and
`
`s- thrombospondin [56. 65].
`
`wntrast to the rather restricted appearance of these
`
`modifications [56]. a fi-linked GIcNAc (fig. So) can
`
`-Mached to an extended array of proteins [6640}.
`
`° far. no elongation of this type of modification has
`
`reported. It is confined to a single monosaccharide
`
`to the protein, and is present on a steadily
`
`_ g list of intracellular proteins. for example cy-
`
`eletal constituents such as erythrocyte band 4.]
`
`.
`-' . neurofilaments, cytokeratins 8. 13 and 18, tran-
`
`option factor Spl or c-myc, estrogen receptor and
`
`-[70, 71]. A reciprocal relationship to phosphoryla-
`
`is assumed for the reversible formation of Glc-
`
`_ fil-Serfl'hr, as such glycosylation sites may also be
`
`ts of protein kinase(s) action. Functional implica-
`5 also include a role in transcriptional initiation by
`
`A polymerase I! and in cytoskeletal assembly and
`
`etion [TO—72]. GlcNAcylation is also possible
`
`hydroxyproline in slime molds. where a GalfFuc-
`
`taining pentasaocharide appears to be the nonim—
`
`ogenic
`product of
`a
`hitherto
`unrecognized
`
`aplasmic O-glycosylation pathway [73]. The lack of
`
`unogenicity is reason to suspect occurrence of simi-
`
`structures in mammals. Hydroxyl groups of amino
`
`. side chains can further accommodate conjugation
`
`'- tb DI-Gal (Ser in plant glycoproteins such as the
`
`
`
`Page 10 of 59
`
`potato lectin}. with e-Glc (Tyr in glycogenin, the primer
`for glycogen biosynthesis),
`fi-Gal
`(Hyl
`in collagen)
`which can be extended to a disaccharide by al,2-linked
`Glc (surprisingly. the presence of this otherwise uncom-
`mon disaccharide O-linked to Thr is also known from
`the surface layer glycop