Pure & Appi. Chem., Vol. 56, No. 7, pp. 859—877, 1984. 0033—4545/84 $3.OO+O.OOPrinted in Great Britain. Pergamon Press Ltd.

©1984 IUPAC

SPATIAL STRUCTURES OF GLYCAN CHAINS OF GLYCOPROTEINS IN RELATION

TO METABOLISM AND FUNCTION1 SURVEY OF A DECADE OF RESEARCH

Jean Montreuil

Université des Sciences et Techniques de Lille I, Laboratoire de Chinie Biologiqueet Laboratoire Associé au C.N.R.S. n° 217, 59655 - Villeneuve d'Ascq Cédex, France

Ab4t'uw.t - Thanks to the progress in chemical and enzymatic methodologies, and tothe use of high-resolution NMR spectroscopy the primary structure of hundredsof glycoprotein glycans has been determined. The knowledge acquired in this fieldhas been decisive in the elucidation of the mechanisms of glycoprotein biosynthesisand of their regulation, and in a better understanding of their biological roles.In this connection, the determination of the spatial configuration became a

necessity. Ten years ago, future results were anticipated by building the Y-shapedmolecular model of a biantenmary glycan. Little by little, this structure has beenrefined and modified thanks to experimental data obtained by using physicalmethods : X-ray diffraction, EPR, NMR, including two-dimensional NMR and one-dimensional 1H—nuclear Overhauser effect (NOE) experiments, neutron scatteringand hard-sphere CXO—anomeric (HSEA) calculations. So, the concept evolvedsuccessively from the Y—, to the T-, the bird— and the 'broken wing"- conformation,until the demonstration that these conformers are interconvertible. The bird-conformation as well as the concept of the mobility of antennae are in a good

agreement with the reactivity of lectins, including membrane lectins, by renderingaccessible any specific sugar structure, and with the activity of glycosyltransfe—rases by making attainable the substituable hydroxyl groups even in the case ofpentaantennary structures. In the same way, we know now that the tetraantennaryglycans adopt an "umbrella conformation" in which the four antennae are disposedparallely to the protein surface and act as protective shields. So could beexplained the resistance towards proteases and the weak antigenicity of numerousglycoproteins as well as the peculiar behaviour and resistance of metastaticcancerous cells since it has been recently demonstrated that membrane glycoproteinsand fibronectin of this kind of cells are significantly enriched in tn- and

tetraantennary glycans.

INTRODUCTION

Ten years ago, in a lecture delivered at the 7th International Symposium on Carbohydrate

Chemistry organized at Bratislava by Stefam Bauer, I reviewed (Ref. 1) our knowledgeconcerning the primary structure, the metabolism and the biological roles of thecarbohydrate moiety of glycoproteins. At that time, the situation was as follows : theprimary structure of about a dozen of glycans had just been determined, the firstinformations about the biosynthesis and the catabolism of glycans of N-glycosylproteinswere available and nothing was known of the spatial conformation of glycans. However, theconcept of glycans as recognition signals was already firmly established thanks to thediscovery by Ashwell's group of the membrane lectins.

Ten years later, it is of interest to draw the general features of our knowledge in thefield of glycoprotein structure and conformation, and to look backward the way which hasbeen followed during the past decade.

For a long time, carbohydrates have been considered as reserve substances, energy-storagecompounds and support structures and regarded as entirely devoided of any "biologicalintelligence". But, during the last 15 years, the chemistry and biochemistry of glycoconju-gates -which result from the covalent linkage of a sugar moiety called glycan with aprotein (glycoprotein) or a lipid (glycolipid)- have acquired an importance as great asthat of proteins and nucleic acid because of the tenacity and trust of a few scientistswho were firmly convinced that the knowledge of the metabolism and of the molecularbiology of glycoconjugates was based on the knowledge of the primary structure of glycans.Thanks to the improvement of chemical, physical and enzymic approaches they developed, thechemistry of glycoproteins progressed rapidly, leading to the determination of the primarystructure of several hundreds of glycans which made possible the knowledge in detail of themechanisms of glycan biosynthesis and catabolism (for reviews, see Ref. 2 to 18).

859

860 J. MONTREUIL

We have now crossed"the shores of the great unexplored continent" announced by A. Gottschalkin 1973 : that of the molecular biology of glycoproteins. We have now to understand thebiological message the glycans carry and to answer two questions. First : why are proteins

glycosylated ?. Second : how act the glycans L) in the protection of the peptide chainagainst proteolytic attack, U) in the induction and maintenance of protein conformation,(U) in the decrease of immunogenicity of proteins, Lu) in the mechanism of recognition andassociation with viruses, with enzymes during the glycan biosynthesis, and with lectins-particularly with membrane lectins, u) in the intercellular adhesion and recognition andin cell—contact inhibition ? Moreover, we know now that the glycan structure of the cellmembrane glycoconjugates is profoundly altered in cancer cells, as revealed by the use oflectins. This molecular transformation could be related to the appearance of surface neo—antigens and could be a factor of cancer induction and metastatic diffusion.

These problems cannot be solved only on the basis of primary structure of the glycans butalso on the basis of their spatial conformation. Unfortunately,up to the 1980's, ourignorance about this latter was immense and our knowledge was essentially restricted to the

structure of the human IgG crystallisable Fc fragment explored by X-ray diffraction byDeisenhofer and Huber (Ref. 18 to 23) . This situation was due to the difficulties encounte-red in obtaining crystals of glycoproteins and of glycans, the X-ray diffraction being atthat time the absolute arm for determining the spatial conformation of molecules. However,in the last few years, our knowledge rapidly progressed thanks to the improvements of theorganic synthesis of glycans which solved the problem of quantity and of purity of thesecompounds, on the one hand, and thanks to the development of the physical methodologies,in particular, of the n.m.r. techniques, on the other hand.

Thus, after a long period of slow development, the research on glycoconjugates suddenlyexplosed during the last decade, simultaneously in the fields of primary structure, spatialconformation, normal and pathological metabolism and molecular biology. In this connection,the following title of a review of Nathan Sharon and Halina Lis (Ref. 10) perfectly reflectsthe situation : 'Glycoproteins : research booming on long—ignored, ubiquitous compounds".

CONFORMATION OF THE PROTEIN MOIETY

Our knowledge of the spatial conformation of glycoproteins as a whole is still too limitedto draw general laws. However, fundamental informations have been brought about theconfiguration of the peptide chain around the glycan-amino acid linkage to which I shallfocuse this short review.

It is now well established that the existence of key peptide sequences called qLLov16

(Ref. 24), around the glycan receptor amino-acid, is a prerequisite feature for glycosyla-tion of a protein. For example, the tripeptide sequence Asn-X—Ser(Thr), where X can be

almost any amino-acid, except proline, is the prerequisite for N-glycosylation of proteins.However, as sequons of certain peptide chains are sometimes not glycosylated, their presenceis not a sufficient condition for glycosylation. Recently, a second requirement for carbo-hydrate attachment to proteins has been defined which is associated with a specificsecondary structure of the polypeptide chain : the -tLUtn. In fact, by applying predictivemethods (Ref. 25) to amino acid sequences adjacent to the glycosylated sites of numerousglycoproteins, Aubert and Loucheux-Lefebvre (Ref. 26 & 27) and Beeley (Ref. 28 & 29)demonstrated that glycans are located in amino—acid sequences favouring turn or loop orhairpin structures (Fig. 1). Most are in tetrapeptide s-turns. These turns consist of only4 amino-acids enabling a polypeptide chain to reverse by nearly 1800, this conformation beenmaintained by a hydrogen bond between the CO function of the first amino-acid of the tetra-peptide and the NH function of the fourth amino-acid. Asparagine, serine and threonine areespecially abundant in these regions.

Thus, the glycosylation by enzymes may be favoured as turn and loop conformations aregenerally located at the surface of globular proteins, making the receptor amino—acidsreadily accessible to glycosyltransferases (Ref. 30). In addition, proofs have been obtainedthat the carbohydrate moieties are located on the outside of glycoprotein molecules. Thisresult is in a good agreement with the role of recognition signals played by numerousglycans. Moreover, it is possible that the protection against proteolytic attack may be dueto the masking of turns and loops by the glycans acting as protective shields. The poorimmunogenic activity often observed with glycosylated proteins could be also related tothis masking. This opinion is reinforced by the molecular model given in Fig. 19.

However, it seems that the presence of the required sequons located in s—turns is not asufficient condition for glycosylation. In fact, the knowledge of the complete primarypeptide sequence of some glycooroteins leads to the conclusion that the double concept ofthe sequon and of the s-turn must be completed by an additional one, because certain

glycoproteins possess residual unglycosylated sequons located in s-turns. For examDle, eventhough the human lactotransferrin peptide chain contains three Asn—X-Ser(Thr) sequons in

Spatial structures of glycan chains of glycoproteins 861

1325

40

•5•0 4

6,N752115 110 105 100

125

130

135 140 145 150

155

160

175 170 165

Fig. 1. Suggested secondary structure of cq—acid glycoprotein (Ref. 26)according to the predictive method of Chou and Fasman (Ref. 25). Residuesare represented in their respective conformational state : helical (o),s—sheet (,-), coil (-), lB-turn tetrapeptide ( ). Asn 15, 38, 54, 75 and85 carry a carbohydrate moiety.

B-turns, only two asparagine residues (Asn 137 and Asn 488) are glycosylated (Ref. 31). Thus,at least a third condition must be envisaged. We hypothesize that the protein could carry

specific peptide sequences recognized by and associating with the glycosyltransferases.

STRUCTURE OF GLYCOPROTEIN GLYCANS

The knowledge we now possess of the structure of numerous glycans raises a very excitingproblem from a comparative biochemical point of view and entirely confirms the conceptsI developed since 1962 (Ref. 1, 32 & 33). In fact, glycan structures are not randomlyconstructed. On the contrary, they are subject to laws the bases of which are found in thespecificity of glycosyltransferases and in a conservative evolution of these enzymes. Inthis connection, the survey of all the glycan structures determined up to now shows that theymay be divided into families, within each of which, structures are very similar and present

common oligosaccharide sequences,whether they originate from animals, plants, microorganisms,or viruses. Consequently a series of classes, concepts and rules is now firmly established,including the sugar-protein linkage.

Nature of linkages between glycans and proteins

Glycans are conjugated to peptide chains through two types of primary covalent linkagesN-glycosyl and 0-glycosyl linkages, leading to the definition of two classes of glycopro-teins : N-glycosylproteins and O-glycosylproteins (Table 1).

TABLE 1. Classification of glycoproteins

Types of glycoproteins Types of linkagesMonosaccharide Amino acid

N-gPyoep/wteLvi4 G1cNAc Asn

O-gLico4 p)o-teAvUMucin type Ga1NAc Ser,Thr

Proteoglycan type Xyl Ser

Collagen type Gal OH-Lys

Extensin type SAra OH-Pro

862 J. MONTREUIL

Until now, the only N-glycosidic bond characterized in glycoproteins is the N-acetylgluco-saminyl-asparagine : GlcNAc(1-N)Asn, which is considered as the ancestral linkage (Ref. 34).on the contrary, the 0-glycosidic type offers a wide variety of linkages the most distribu-ted ones being the following-L - linkage between N-acetyl-D-galactosamine and L-serine or L-threonine : GalNAc(z-1,3)Seror GalNAc(c-1,3)Thr, alkali-labile, found in very numerous glycoproteins which are said ofthe 'mucin type"Lci - linkage between D-xylose and serine : Xyl(1-3)Ser, also alkali-labile involved in theacidic mucopolysaccharide-protein bond of proteoglycans

- linkage between D-galactose and 5-hydroxy-L-lysine : Gal(1-5)OH-Lys, alkali-stable,characterized in collagens

- linkage between L-arabofuranose and 4-hydroxy-L-proline : L-Ara(1-4)OH-Pro, alkali-stable, identified in plant glycoproteins.

Primary structure of glycoprotein glycans

1. The concept of the common inner-core. The carbohydrate moiety of N- and O-glycosylproteinsderive from the substitution of oligosaccharide structures common to all glycans of a givenclass of glycoproteins. These non-specific and invariant structures are conjugated to thepeotide chain and constitute the ,LflneA-cO/ie. The most current inner-cores characterizeduntil now in glycans are described in Fig. 2. Cores A and B exist in the O-glycosylproteins

Gal(1-3)GalNAc(ct1-3)Ser or Thr AG1cNAc

(1_3)\/Ga1NAc(cZl-3)Ser or Thr B

GlcNAc(1—6)

Gal(1—3)Gal(1—4)Xyl(1—3)Ser C

Man(z1_3)\

)Man(1—4)GlcNAc(1_4)GlcNAc(1_N)Asn D

Man(od-6Y'

Fig. 2. Oligosaccharide inner-cores of glycoprotein glycans.

of the mucin—type. Core C constitutes the terminal sequence of almost all the glycosamino-glycans of proteoglycans. Core D is common to all N-glycosylproteins.

2. The concept of the antenna. The glycan structures derive from the substitution of theinvariant inner-cores by a wide variety of oligosaccharidic structures which carry thespecificity of the glycans and constitute the variable fraction of these latter. On thebasis of their morphology, their flexibility and their property of being recognition signalsthe term i,tevtviae has been proposed, from 1974, for the outer arms substituting the inner-cores (Ref. 1).

3. Glycan structures present in O-glycosylproteinsGyavzís conjiigwted thiwuh a Xy31-3)Sex LLnfzctga. This group of glycans consists of theacid mucopolysaccharides or glycosaminoglycans. All are linear polymers made up of disaccha-ride repeating units (Fig. 3) and 0-glycosidically linked to the peptide chain in the

GalNAcU31—4)GlcUA(31—3)

SO3-

Fig. 3. Primary structure of seryl-chondroitin 4-sulfate.

so called proteoglycans, except in two cases : -L) that of hyaluronic acid which does notseem to exist as a proteoglycan -U) that of keratan sulfate I which is conjugated to theprotein through an N-acetylglucosaminyl-asparagine linkage.Gcctn ojaga,ed tIvwagh a GaNAcc1-3)Se,'i 0/i TIVt LLntaaga. This group mainly comprisesstructures consistingofmucins and these glycans are often designated as nac,Ln—tL(astructures. Some of these structures are described in Fig. 4. The main observation whichcould be formulated about these structures is the lack in structural specificity ofnumerous of them. In fact, the same structures can be found in glycoproteins of verydifferent origins and roles. So, they could be regarded as structures playing essentiallya role of a physicochenical order and intervening essentially in the conformation of thepeptide chain. In this connection, it is worthwhile to note that this kind of glycans isoften located in "strategic" domains of the protein moiety, for example, in the hinge regionof immunoglobulins.

Spatial structures of glycan chains of glycoproteins 863

Ga1(1-3)GalNAc(ct1-3)Ser or Thr 1NeuAc (ct2-3) Gal (1-3) Ga1NAc (czl-3) Ser or Thr 2

Gal (131—3)

)Ga1NAc (al-3) Ser or Thr 3

NeuAc (ct2-6)"

NeuAc (a2—3)Gal (1_3)\

)GalNAc(c1-3)Ser or Thr LI

NeuAc (a2—6(

Fuc(c1—2)Gal(1—4)GlcNAcU31—3)\)GalUBl-3)Ga1NAc(al-3)Ser or Thr 5

Fuc (cd—2)Gal (1—4) G1cNAc (1—6)

Gal (1—4)GlcNAc (1_3)\

)GalNAc(cal-3)Ser or Thr 6

Gal(1—4)GlcNAcU31—6(

Fig. 4. Some examples of structures of glycans 0-glycosidically conjugatedto protein through the linkage GalNAc(c1-3)Ser or Thr. These glycans aredistributed in the following glycoproteins : Structure 1 : anti-freeze

glycoprotein from antarctic fish, human chorionic gonadotropin s—subunit,human serum IgA1, epiglycanin of TA3-Ha cells, T-reactive erythrocytes,rat brain glycoproteins. Structure 2 : human glycophorin, bovine kappa-casein, fetuin, human milk secretory IgA, N—blood group. Structure 3submaxillary mucins, rat brain glycooroteins, trout and herring eggs.Structure 4 : human glycophorin and gonadotropin s-subunit, fetuin,lymphocyte plasma membrane, M-blood group. Structure 5 : porcine blood—groop H substance. Structure 6 : bronchial mucin of patients sufferingfrom cystic fibrosis. For reviews, see Ref. 9 & 14.

4. Glycan structures present in N-glycosylproteins. The N-glycosylproteins are divided intothree families according to the nature of the carbohydrate linked to the pentasaccharidicinner-core D (Fig. 2). In the first family, the glycans contain mannose and N-acetylgluco-

samine only. They are called glycans of the oLLgomannoS-LdAic 4jpe or hLgh-rnctnvws typeglycans. In the second family, the sugar composition of glycans is more complex. In fact,these glycans contain galactose, fucose and sialic acids in addition to mannose and N-acetyl-glucosamine. They derive fundamentally from the addition to the pentasaccharidic inner-coreD of a variable number of N—acetyllactosamine residues : Gal(1—4)G1cNAc. These structureshave been called glycans of the N-cae)tyP1ctC)tO4CinLne type. or of the eOrnpeX. type. In thethird family, the glycans simultaneously have structures of the oligomannosidic and of theN-acetyllactosaminic type. They belong to the type. or

hybzLd type. glycans.

G4jcctvi4 o th oLLqornctvivio-Ld,Lc tpe (,Lqh-mcu'ivto type). The glycans of the oligomannosidictype constitute a very homogeneous group. In Fig. 5 are given the structures of the two

Man (c1-2)Man(c1-2)Man(a1-3)

Man(c1—2)Man(a1-3) ,,Man31—4)GlcNAc(131—4)GlcNAc(1—N)Asn 1132 A / 3 2 1= = Man(C1-6) = = =

Man(ml_2)Man(al-6) '

Man(ct1-3)Man

(al_3\)Man(i1—4)GlcNAci31—4)GlcNAcU31—N)Asn 2

jMan(c1-6)"Man (o1-6)

Fig. 5. Structure of glycans of the oligomannosidic type (high-mannosetype). Structure 1 is present in calf thyroglobulin unit A, human IgD andmyeloma 1gM, Chinese hamster ovary cell glycoproteins, bovine lactotrans—

ferrin, soybean agglutinin, Newcastle virus, scorpion hemocyanin.Structure 2 has been found in Taka—amylase, in hen ovalbumin and

in human myeloma 1gM. For reviews, see Ref. 9 & 14.

864 J. MONTREUIL

glycans, the most widely distributed in plants and animals. These structures are common to

numerous glycoproteins of different origins and roles and, thus, do not present anystructural specificity. On the basis of the findings concerning the biosynthesis of theN-glycosidically linked glycans, they could be regarded as metabolic intermediates in thematuration of the glycans of the N-acetyllactosaminic type as shown in Fig. 6 (For reviews,see Ref. 7, 9, 10, 12, 14-16 & 35). However, this cannot be considered as their only role.

Glc (al—2)Glc (al—3) Glc (al—3)Man(al—2)Man (a1—2)Man(a1—3)

Man(al-2)Man(al-3)1 )Man(1-4)-R 1

)Man(a1-6)"

Man (al-2)Man (ctl-61

m-gcosLdcseo

Man(al-2)Man(al-2)Man

Man(a1-2)Man(a1-3) ,Man(131-4)-R 2Man(B1-6)7

Man (o1-2)Man(al-6)

ny-i, 2-manno&Ldae,6

Man(al-3)

Man(1-3)1 ,Man(1-4)-R 3

Man (ml-6/

Man(ml-6)'

tJ-ag&LaoainLny-t/LcLn iS VLa6 I

G1cNAcU31—2)Man(cxl—3)

Man(a1-3)Man(131-4)-R 14

Man(cd-6)

Man (ctl-6)

GAc-dependvito-rncLvrno4LdW5 e

GlcNAc(131—2)Man(al—3)

an - -RMan(ml-6)

t/Lans 15 euL6 I I

GlcNAc(f31-2)Man(O1-3)

,,,Man(131-4)-R6

GlcNAc()1—2)Man(ct1—6)

EongatLo n cixtLo nscatcthjzd by gataoy!-and La4iLtJLano 15e/ao eo

Biantennary glycan of the N-acetyllactosaminic type(Structure 2 of Fig. 10)

Fig. 6. Biosynthesis pathway of a glycan of the N-acetyllactosaminic type.R = GlcNAcU31-4)GlcNAc(31-N)Peptide. Glycan 1 is transferred en bloc froma dolichol pyrophosphate intermediate onto the nascent peptide chain inthe granular endoplasmic reticulum. The newly formed glycoprotein isfurther trimmed in the Golgi apparatus by specific ct-glucosidases and -mannosidases leading to glycans of the oligomannosidic tvoe first to theglycan 2 and then to the glycan 3. This latter is the acceptor for thetransfer of the N—acetylglucosaminyl residue on the c-1,3—linked mannose(glycan 4). The two o-1,3 and c-1,6 "extra" mannose residues are thencleaved off leading to the glycan 5 which is the acceptor of the N-acetyl-glucosaminyl residue on the -1,6-linked mannose (glycan 6). The completion

•vT 6 •;i s 'sMeTAei 104 UWnqPAO uq UT : Nbi U1OTeJUI Uuinq pU UTSQOpO4t eUAOq UT : ! UTsdopoipt eUAOq

UT : : peZT[DIT4D dA ptqq eq ;o SUPDAT ;o enonis 6 bT ,(9—Tx) UPH

(9—I?O)UPw ,// l(E-T3)UPw

(—TDDvNT \\ (-Tn)UPN

'ft—is) YNDTo(i—I) TE

1(9-pO) UW (9-pD) UN

3 (E—T?o)Uw(—I)ovNDTD

(9-JD) UW I US(N-I) DVNDTDft-T) DNDi ft-T) UW

et; o sfuoeq srnu; UP ( 6 bTI) edA DTUTS0DPTTA3-N -T ;o pUP DTPTS0UUU10TIO T: ;o onnxs UOSeid 1TSfl08UTflmTS SUPDATb qD4M UT SUTOO1dTASODAT-N ;o dno2b p1Tq

0 UOTTUT;ep eq o p sepTddoDA-[b UuxnqAo U0 sbUTpUT pjqPn 9o frj-j (j 30u) UTO01dODIcTb TT AIOAO 10SUXPH OSOUçq3 pUP (Uv JI) UT SU1ODPT uouinq

'(T J)J) OUqUXOUI ADO1qA1B UT pezTle DP1PD uoeq eApq sopTded-(TASooATb) Atod pourou

( dflO1f) °YNDT(J7—T)TPE { (T0) rU } (E-T3)T 10 (cJ dnolD) °YNDTO(-T)TPD {(_Tfl)°fl } 10 (0 dnolD) °YNDTO (—T) TPD (—Tfl) Dfl 'DVNDTD (f7—Tg) TPE (g—o) VflN DYNDID

(-T)I(E-n)oYnoN = 'eUPlqUioul OADO1qA1e UPWfl UI USY(N-J)DYNDT (T-TDoYNDT = I T O tUO1 SOTXPA U qDqM UT SepTded-(TAsoTb)

A1od oq ;o sUPDITb i1PUUeUPq 04 O O1flDfl1S DçSPq 8 T[ U (9—T0)UPN(—T)oYNDTEft—Ti)TPs (—Ti)3YNOTEft—IDTPo —T

I_ft_g)Up/ U (—IDovNoTft—T)TPs —t

'(8 bT) SODUOnbOS TUTtUPS0DPITJeDP—N—AT0d 10 —ObTIO 100UTI bUTUTPU0D SO1flDfl1S '1OAOO1OW U0TPTIcS0DPIPb-U0U 0 IPUTS P 0 10 SBSP1eSUP11AS03P1Pb UT [°T o o enp AqPqo1d

UPDAT6 UT110SUP10A0 Ueq 0 01flDfl14 L bT

(9—TUI)UPW(—1DDvN3TE

USY(N-1) DVNDT ft-1) DYNoTDft-g) UPW/ ft-1) DYNOTE \ 1—1nvNoT

\E-1V) UPW

OYNOT

'U. bc) U110SUP10A0-UOq ;o SUPDAT UT SO 'eUTTfl0 UT 01PS A1UO ST SOflPTSO1 0UTUIPS0OPTTA0DP—N 9t4 30 U0TPtU103 0T UP O0TdU103UT PIP SbUT4DUP1q 'ATTPUOTSPDOO

(O 3U UT MOTAPI) 050003 UP (6 0 8E 30 UT SMOTAO1) OTTPTS T 'TPUbTS U0TTUb0001 SO W0T S0PUbTS0p U0TTS0d IPU10X0 IcTTPoTPtuoSAS 05014M SOpT1Pq300SOUOUI 0M

30 0DUP10dU1T 1PTfl3T1Pd oq 0U[10pUfl 01 UT peq1osep S01flDfl1S eq 'U0T000UU03 STq UI STPUbTS UoTTUoDo1 So iPU0SS0 oo oq oq Sseqodq eq S100AP3 U0PA1oSq0 STq

PUP S01fl0fl1S 30 0T1PA PPTM P cuese1d odA 0TUTmPS03PTTA0DP-N eq ;o SUP0I[b 'suooA1b

p0q1050p 0A0qP oq 0 SP13U00 UI ( dh xdwo) c)h' D114 Wvc'o-vflhpDv-N "P- Yo c'14VD&

(Li 0 30 POS 'SMOTAOl 10) OUP1qtUOU PUISP1d PUP T5T UT U0S01d S10d0001 30 U0TTUb0D91 OT3TOOdS Aq SOUIOSOS1c1 0 SOUIAZUO IPUOSOSIcT p0ZS0qUUcS AIMOU 0b1P adA

OTPTSOUUPUIOOTTO 0qU 30 SUPOIT6 paPA10qdS0qd 'lPAOPlOW SPPTSOUUPUL&IOd UP -ObTT0 103 'T -T3TOPdS P tTM PT10DPq 0Lt 30 )7YCI 0L U0 U0S01d UT00T P 30 SUPPU Aq 'U0TD03UT UP U0T

-PZTUOTOO 103 aTsTnbe1e1d 'S--eo --jeqyçde UPUIUX4 o spirçq ''QD 'e1duxoxe 10 STPU5TS U0TTUb0D01 S01fl13fl15 DTPTSOUUPUIO6TTO pq M045 SUOflPA1OSqO OUXOS '4003 UI

(6 5TT 30 se1rtonqs 005) edk p1qcq eq 30 T UT901d -00T oq ' UPDATO Pq 30 0UP1 UPW-'1--?3 04 U0 1c1U0 SdOTPAPP T 31

• (S Ti1 30 UP V S01flDfl1S oos) od DTpTsoUUPaIoTTo 3° T UTa01d -ODA1b Pq ' 10 SUPOAT5 30 TOAPT eq o sdos ssoooid U0TcP1tiPUI eq 31

SPOPTSP1 UP TiSoon; '1Asoop1b bUTPPP Aq pPAPTqDP 5T UPqo 9L[ 30

S98 suT0o1doJJT jo suqo o S nirii 1PpPdS

866 J. MONTREIJIL

NeuAc (a2—6) Gal (1—4) G1cNAc (1—2)Man (al—3)\

,Man(1—4)G1cNAc(1—4)G1cNAcU31—N)AsnMan(al-6)"

NeuAc (ct2—6) Gal (1—4) GlcNAc U31—2)Man(al—3)\

')Man(1—4)GlcNAc U31—4)GlcNAc (1—N)Asn 2/3 2 1NeuAc(a2—6)Gal(I31—4)GlcNAc(1—2)Man(a1—6) = = =

NeuAc(ct2—6)Gal(131—4)G1cNAc(131—2)Man(al—3)\G1cNAc ( 1—4) )Man (f3 1-4) GlcNAc ( 1—4) GlcNAc ( 1—N) Asn 3

NeuAc(a2—6)Gal()3l—4)G1cNAc()31—2)Nan(al—6)' Fu6NeuAc (ct2-6)Gal ()31-4) GlcNAc (1_2)Man(a1_3)\

,>Man(131—4)GlcNAc(131—4)GlcNAc(131—N)Asn 4Gal(1-4)GlcNAc(1-2)Man(a1-6) Fu6

(al—3)Fuc

NeuAc (a2—3)Gal (1—4)GlcNAc()31—4)

1Man(a1_3)NeuAc(a2—6)Gal(131-4)G1cNAc()31-2)' >Man31—4)GlcNAc()31—4)GlcNAcW1-N)Asn 5NeuAc (a2—6) Gal ()31—4)G1cNAc U31—2)Man(al—6Y'

NeuAc (a2—6) Gal (131—4) G1cNAc (131—2)Man(al—3)\

NeuAc(a2—6)Gal(131—4)G1cNAc(131—2)/ManU3l_4)GlcNAc(131_4)G1cNAc(131_N)Asn 6

1Man(al-6)NeuAc(a2—3) Gal (131-4)GlcNAc (131_6)I

A NeuAc(a2—3 or 6)Gal(f31—4)G1cNAcU31—4)

1Man (cd—3)B NeuAc (a2—3 or 6)Gal (131—4)G1cNAcU31—2)1 Man(f31—4)G1cNAc (131—4)GlcNAc()31—N)Asn

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Gal (al—4)Gal (131—4)GlcNAc (131—4)-Man(a1—6)

Gal (al—4)Gal (131—4)G1cNAc (131—6)'

Fig.1O. Structure of glycans of the N-acetyllactosaminic type (complextype). 1 : monoantennary glycan of secretory component from human milk andof human chorionic gonadotropin; 2: biantennary glycan of human serumtransferrin ; 3 : monofucosylated and bisected (presence of a bisectingN-acetylglucosamine residue) biantennary glycan of human IgG ; 4 : difu-cosylated biantennary glycan of human lactotransferrin 5 and 6 : tn-antennary glycans of human serum transferrin ; 7 ; tetraantennary glycanof human a1-acid glycoprotein ; 8 : pentaantennary glycan of turtle—doveovomucoid possessing a P1-serologic activity. For reviews, see Ref. 9 & 14.

h1jbLd type. These structures are considered as intermediates in the biosynthesis of theglycans of the N-acetyllactosaminic type (see Fig. 6).

ConclusionThe survey of the structures we described shows that the substitution of monosaccharidestaking part of a glycan structure conforms to a certain orthodoxy which is very restrictive,the basis of which is found in the specificity of glycosyltransferases and in a conserva-tive evolution of these enzymes. In fact, the possibilities of substitution of a given sugar

Spatial structures of glycan chains of glycoproteins 867

are limited to 1,2 or 3 well—defined monosaccharides, generally conjugated by a uniquetype of glycosidic linkage. So could beestablished the concept ofone-glycosidic bond-one

glycosyltransferase' , the action of which is, in addition, guided by substitution ruleswhich depend on the primary structure and conformation of the nascent glycan (For reviews,see Ref. 14 & 35) . Moreover, the substitution rules raise very interesting questions ofcomparative biochemistry and of phylogeny and, in addition, allow the foundations to be laid

for genetics of glycosyltransferases.

SPATIAL CONFORMATION OF GLYCANS

Glycans of O-glycosylproteins

Glycosaminoglycans and proteoglycans. Conformation of acidic mucopolysaccharides and assem-bly of proteoglycans are pretty well known due to their relative simplicity (for recentreviews, see Ref. 44 & 45) . X-ray diffraction studies have shown that the polysaccharidesare generally in a helical conformation. For example, X-ray diffraction of highly crystal-line oriented fibers of hyaluronic acid demonstrates that this compound possesses a doublehelical structure in which two identical left-handed strands having four disaccharide unitsper turn, are antiparallel to one another and maintained in this conformation by hydrogenbonds. In the intercellular matrix of cartilage, most of the proteoglycans exist in theform of aggregates which are constituted of the association of proteoglycan monomers withhyaluronic acid. The structure of the monomer molecules is that of a central core to whichglycosaminoglycans are covalently bound. A low molecular weight (43-48 Kd) glycoprotein,called £AivLta pJw;teAIvL is responsible for promoting proteoglycans-hyaluronic acid aggregation.

Whether this kind of architecture could be generalized and would exist in the intercellularmatrix of any tissue remains to be demonstrated.

Mucin-like glycans. In contrast to proteoglycans, very little is known at the moment aboutthe conformation of 0-glycosidically linked glycans and of O-glycosylproteins, except inthe case of submaxillary mucins. In fact, mucins have an elevated axial ratio and are in arod- conformation maintained as a result of repulsive electronegative charges carried by thesialic acid residues present in the 800 disaccharide moieties NeuAc(n2-6)GalNAc(cd-3)Ser orThr which are almost regularly distributed along the long peptide chain (Ref. 46) . Thisextended conformation is responsible of the high viscosity of mucin solutions and of secre—tions of mucous cells. Removal of sialic acid makes the glycoprotein globular, the solutionsof which become weakly viscous. This example clearly illustrates the role the glycans couldplay in maintaining the protein in a biologically active conformation.

Glycans of N-glycosylproteins

1. From the Y- to the "broken-wing" conformationThe Y-conjço/unwtAion. Until recently the only image we had of the spatial conformation ofglycams N-glycosidically conjugated was speculative because it was obtained by the construc-tion of molecular models (for review see Ref. 1, 9, 14, 17 & 47). The first one was presen-ted (Ref. 1) in 1974 at the 7th International Symposium on Carbohydrate Chemistry inBratislava. The construction of the biantennary glycan molecule of human serotransferrin

(Fig. 10; structure 2), in creating thermodynamically possible hydrogen-bonds, led to they- shaped conformation illustrated in Fig. hA. This structure may be divided into two parts.The first is compact and constituted of the Lnv pemtasaccharidic innercore of mannotriosido-di-N-acetylchitobiose. The terminal trisaccharide Man31-4)GlcNAc(131-4)GlcNAc(IB1-N) is flat(Fig. 12) and rigid due to hydrogen bonds. The second part is looser and made up of the twoantennae constituted of the trisaccharide NeuAc(a2-6)Ga1()31-4)GlcNAc(31-2). It correspondsto the VcVL structure attached to the inner-core and carries the biological activity.

Of course, when I proposed the Y— shaped model, I had the choice between different confor-mations, but I preferred the Y-conformation because, at that time, one still believedthat lectins, including membrane lectins discovered in 1968 by Morell and Ashwell ( forreviews, see Ref. 48), were able to recognize and to bind only the monosaccharides in aterminal non-reducting position. Thus, the Y-conformation was in a good agreement with this

concept.

The T-conokrnwtLovi. During the past 10 years, the Y-conformation was revisited and little bylittle refined on the basis of experimental data . For example, using X-ray diffractionstudies, we brought in 1978 (Ref. 49) the first modification and proposed the T-coviouaatLon(Fig. 11B) in which the a-1,3 antenna is disposed perpendicularly in relation to the n-1,6antenna. In fact, the X-ray diffraction pattern of crystals of the trisaccharide Man(ml-3)Man()31-4)G1cNAc isolated from urine of mannosidosis showed that the a-1,3 linked mannoseresidue was perpendicular to the plane of the disaccharide Man(31-4)GlcNAc (Ref. 50).Moreover, the obtained results confirmed that the latterdisaccharide was planar.

This particular position in space of the a—1,3 mannose residue has been experimentallyconfirmed (Ref. 51 to 54). Moreover, according to Sutton and Phillips (Ref. 54),

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870 J. MONTREUIL

3°) crystallographic structural studies of Fc fragment from human IgG (Ref. 19, 20-23, 54 &55) are in favour of an extended conformation of glycan residues.4°) More recently, Bock e,t aL. (Ref. 51) explored the most favoured conformations inaqueous solution of synthetic oligosaccharides related to the N-acetyllactosaminic typeusing high-resolution NMR (both 1H and 13C) combined with hard-sphere ex0-anomeric (HSEAmethod) calculations. The results they obtained indicate that the N-acetyllactosamine unitsare arranged in such a way that they are widely separated in space.

8/wf7evL-wLvtg conwtmwtLovt. On the basis of NMR experiments (Ref. 11, 53 & 64) or by using theHSEA-calculations (Ref. 65), authors proposed the model described in Fig. liD, I suggestedto call bo!zen-wLng conoiunatLon. In this utmost conformation which takes advantage of thelarge angle of rotation of the c-1,6 linkages (see below), the m-1,6 antenna folds backtowards the protein backbone and sets along the inner-core. According to carver et a&.(Ref. 64) this conformation is favoured by a bisecting N-acetylglucosamine residue.

Con4o'wiatLovt o gctns o -the. otLgornctnno&LthLc tqp. Spatial conformation of glycans of theoligomannosidic type has been explored by high-resolution NMR by Vliegenthart €t a!. (Ref.13 & 66). On the basis of the NMR-spectral features, the authors proposed that the favoredconformation of the glycan with 9 mannose residues (Structure 1 ; Fig. 5) is as depicted inFig. 14. In this model, the orientation of the entirely rigid 4-c-D1 trisaccharide antenna

Fig. 14. Molecular model proposed for the oligomannosidic type glycan thatcontains 9 mannose residues. Numbers and letters correspond to the codingused in structure 1 of Fig. 5 (Ref. 13 & 66).

with respect to the Man(1-4)GlcNAc()31-4)GlcNAc(1-N)Asn core is similar to that of the 4-5unit in N-acetyllactosaminic type structures. Regarding the positional arrangement around —the 1,6-linkage of mannose-4' residue, the two disaccharide antennae D2-A Man(al-2)Man(cXl-3)and D3-B Man(o1-2)Man(c1-6) are in an extended-conformation and the -i-p2 part of themolecule is in close proximity of the di-N-acetylchitobiose unit and folds back towards the

peptide chain.

2. Are extended conformations compatible with the properties and biological behaviour of

glycans and glycoproteins ?lnteJLctc,tLon wAik £eetth4. As I mentioned above, the extended conformations of glycans seemto be far from the concept of the terminal monosaccharides as recognition signals. But, weknow now that lectins could recognize and bind "laterally", and not 'terminally", mono— oroligosaccharides in an LntAna,e position. For example, as demonstrated by Kornfeld andFerris (Ref. 67), the most active part towards comA of an N-glycosidically linked glycan isnot constituted of an a—mannose residue in a terminal position, but of the disaccharideGlcNAc(1-2)Man(m1-3 or 6). Thus, the glycans in an extended conformation provide goodtargets for recognition by other proteins in general, by lectins in particular.

AacebLLL4j to eJIZyneS. The extended conformations are the most satisfactory ones from thepoint of view of the glycan biosynthesis because all smbstitutabie hydroxyl groups arereadily accessible to glycosyltransferases, as shown by the following examples

1°) Substitution of c-4 of the )3-mannose residue by a bisecting N-acetylglucosamine,like inhuman IgG (Fig.1O ; Structure 3). This residue interacts with the two ct-mannoseresidues 4 and 4' and covers in particular a wide area of the surface of G1cNAc(iBl-2)Man(cti-3)Man(31-) unit (Ref. 57 & 64), thus explaining -L) the shifts of the H-i of the threemannose residues observed in NMR (Ref. 11 & 18) ; ü) the non-reactivity with con A of thiskind of glycans because of the steric hindrance induced by the bisecting N-acetylglucosamineresidues LU) the inhibition of certain glycosyltransferases (Ref. 35 & 68).

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Spatial structures of glycan chains of glycoproteins 873

So could be explained the resistance of certain glycoproteins and their weak antigenicity,glycans acting as protective shields. Moreover, the umbrella conformation of glycans issolidly maintained by ionic bonds between the electronegative charges of sialic acidresidues and electropositive charges of basic amino acids, as demonstrated by the small-angle neutron scattering study of Li t aL. (Ref. 71). Removal of sialic acid residues,by making the antennae free and consequently mobile, abolishes the protective effect ofglycans, so rendering the protein more antigenic and accessible to proteases.

If such structures are present in viral envelopes, one can appreciate at what level theviruses have brought the art of the camouflage and understand how the glycans may play arole in masking or altering the nature of the protein's antigenic sites and, hence, playa role in the process by which the viruses could escape recognition by the immune system.The possibility that glycans modulate recognition of viral proteins by the immune systemis discussed in a paper of Wiley t al. (Ref. 73).

The protective effect that play the glycans towards the protein moieties could also explainthe peculiar behaviour and resistance of metastatic cancerous cells since we know now thatthe membrane glycoproteins of cancerous cells are significantly enriched in tn- and tetra-antennary glycans as demonstrated by several authors (Ref. 58, 74-80). Table 2 clearlyillustrates this fact. Moreover, the new glycan structures carried by the metastatic cellscould explain the tissue specific localization of the latter by specific lectin recogni-tion.

TABLE 2. Increase of the proportions(*)of tn- and tetraantennary glycans fglycoproteins in the case of virus-transformation of BHK 21/C13 fibro-blasts

Branching type of glycans Biantennary Tn + tetraantennary

"Normal" BHK 21/C13 69 31

HSV-transformed BHK 21/C13 32 68

(*) Proportions are expressed in per cent

In the same way, Delannoy at aL. (Ref. 81) recently observed that fibronectin from cancerouscells in culture is enriched in tn- and tdtraantennary glycans leading to a completeinversion of the ratio biantennary/tri + tetraantennary structures (Table 3 ). This trans-formation is accompanied by an increase of the sialylation. The profound modifications weobserved could be related -L) to the loss of reactivity of the protein moiety of the fibro-

nectin by masking the lectin—sites, £L)to the disappearance of fibronectin from canceroustissues and LU) to the mechanism of metastasis itself.

TABLE 3. Increase of the proportionsof tn- and tetraantennary fibronectinglycans and of the degree of sialylation associated with the viralmalignant transformation of BHK 21/C13 fibroblasts.

8/LcLnahLng tpe o gftjaavis Bntenncv TL + eaanLerznatj"Normal" BHK 21/C13

HSV-transformed BHK 21/C13

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40

72

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HSV-transformed BHK 21/C13

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45

25

14

33

4

23

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b — Glycan and protein conformation. The concept of the extended conformation ofglycans allows a better understanding of the role the carbohydrate moiety plays in the conforma-

tion of proteins through specific protein-carbohydrate and carbohydrate-carbohydrate interac-tions.

A very fine demonstration was recently brought by Sutton and Philips (Ref. 54) by using X-raydiffraction. It concerns the Fc fragment of rabbit IgG molecule which derives from theclassic association of two half-molecules, each carrying a glycan residue of the biantennaryN-acetyllactosaminic type. The latter is in an internal position in the CH2 domain and itsintegrity is indispensable for the maintenance of all properties of immunoglobulins. The

874 J. MONTREUIL

above mentioned authors have demonstrated that (Fig. 20)

Fig. 20. Glycan structure at the Asn—297 residues (indicated 297 and 597)with the backbone of polypeptide chains 1 and 2, in the CH2 domains ofrabbit IgG (Ref. 54).

-ii) the pentasaccharidic innercore lies close to the surface of CH2 domain and interactswith Phe-241, Val—264 and Asp-265 residues-U) the two antennae of each glycan are in an extended conformation, the orientation of thec-1,3 antenna being T—shaped and invariant-<:LL) the a—1,6 antennae of each glycan make several contacts, mainly hydrophobic, with aminoacid residues on the surface of the protein, principally with Phe-243, Pro-246 and Thr-260;Lv) on the contrary, the a-1,3 antennae have no contact with the surface of the CH2 domainbut do interact with each other between the G1cNAc-5 residue of the one chain and theGlcNAc-2 and Man-3 residues of the other chain, and it therefore appears that they areresponsible for maintaining the disposition of the two CH2 domains.

Concerning the interactions between glycans and hydrophobic amino acids, it is of interestto note that the terminal trisaccharide of the inner-core is relatively hydrophobic because

of the presence of the two N-acetylglucosamine residues carrying acetamido groups and ofthe two- and sometimes three-fold substituted -mannose residue. The hydrophobicity of thetrisaccharide is still more enhanced when the N-acetylglucosamine-1 residue is fucosylated.Thus, it is not unreasonable to presume that this hydrophobic part of glycan could interactwith hydrophobic regions of the protein moiety. Its flat structure (see Fig. 12) could berelated to this property.

3. Conformational changes of glycans. Mobility of the antennae.Glycan structures must not be considered as rigid conformations, because when they havea certain degree of freedom, for example, when they present no carbohydrate—carbohydrateor carbohydrate-protein interactions as it is the case for glycans or glyco-oligopeptidesin solution. In fact, glycans present points of flexibility leading to a relative mobilityof the antennae due to the rotation of the rigid pyranose rings around the two bondsattached to the glycosidic oxygen. The rotation is limited in the case of 1 -*2, 1 --3 and1 --4 linkages, thel -3-linkage being considered as the most rigid one. On the contrary, the1 -6 linkages are more flexible because of the additional rotational freedom.

In the case of biantennary structures of the N-acetyllactosaminic type (Fig.1O ; structure2), the most important point of flexibility is located at the ct-1,6 bond linking the manno-se—4' residue to the mannose-3 residue explaining that the Y-, T-, bird- and broken wing-conformers are interconvertible as demonstrated by the following series of experiments

Low anqP X-Jicui at,'ivtg ctvid tez actwt o a LcLaL g?4aoconj ate1. Gallot e aL.(Ref. 59) showed that the glycan moiety of synthetic glycopeptides in aqueous solutionadopts a bird- or a Y-shaped conformation depending on the molecular weight of thepolypeptide block and on the concentration of the glycopeptide, molecular weight of thepolypeptide between 2,000 and 3,500 and high concentration favouring the Y-conformation.

In the same way, Gallot et aL. (Ref. 82 & 83) synthesized amphipatic liposaccharides bycoupling glycoamino acids with fatty acids containing from 8 to 24 carbon atoms. Mixturesof these glycolipids and of phospholipids lead to the formation of long and parallellamellar structures, glycans and water filling the space between the layers. As long as the

CH2

Spatial structures of glycan chains of glycoproteins 875

glycolipids are in small quantity in the ternary system glycolipid/phospholipid/water, theglycan takes up a bird-conformation. When the quantity is high, it becomes Y-shaped.

Eeex,tiwvi paJtcLmcLgvLet(ic )Leovlctvice oj pLn-&tbe11ed gctno. The internal degree of freedom ofrotation in space of the antennae has been pointed out by am electron paramagmetic resonancestudy by using spin labelled bi- and triantennary glycans (Ref. 84) . Spin-spin interactions

originating from collision effectabetweem probes have been demonstrated. The interactionswere higher in the case of triantennary structures. The addition of ComA reduced the mobili-ty of the probe bound to the biantennary glycams which are able to bind ComA. It does notaffect the spin-labelled triantennary glycanswhich are well known to be unable to associate

with ConA.

SnictU-w'ig viewtiiovi cattvuinq stud ohuincrn aj-ac,Ld gegcopwte,Ln has shown that also inthe case of tetraantennary structures all antennae are mobile and able to adopt an'umbrella-conformation" (conformer (c) of Fig. 17) or a "reversed umbrella-conformation"

(conformer (b) of Fig. 17) (Ref. 71).

4. Concluding remarksThe above data bring out the fundamental role that plays the mannotriose core in theconformation of glycans of N-glycosylproteins. In this connection, it is amazing to notethat all "strategic" branching points systematically associate a rigid 1,3-linkage with amobile 1,6-linkage as shown in Fig. 21. What is the reason for the relatively invariant orien-

Man(m1—3) GlcNAc(1_3)\

''Man- A "Gal- B

Man(ctl—6) GlcNAc(1—6)

Gal(tBl—3) GlcNAc(1—3)

GalNAc- C Ga1NAc- D

GlcNAc(1-6) GlcNAc (11—6)

Fig. 21. "Strategic branching" associating 1,3- and 1,6-linkages. A : inner-coreof N-glycosidically linked glycans B and C : branchings currently found in 0-

glycosylprotein glycans of the mucin-type ; D : inner-core of Ii active glycans

from sheep gastric mucin (Ref. 85).

tation of a T-shaped 1,3-bond with a highly flexible and mobile 1,6-bond ? Probably inorder to favour, when necessary, an extended conformation of the antennae which favours

itself L) the occupation of their "vital space" by the glycans U) the interaction of the

glycans with their own protein ; LLL) the glycan-glycan interactions Lv) the action of

enzymes ; v) and/or the recognition of glycans by receptors.

CONCLUS IONS

Ten years ago, I wrote as a conclusion of my report entitled "Recent data on the structureof the carbohydrate moiety of glycoproteins. Metabolic and biological implications""At the end of this brief review, we can conclude that the problem of determination of theprimary struture of glycans is virtually solved. However, it remains to miniaturize theprocedures in order to extend their application to very small quantities of biologicalsubstrates.Thus, we can look forward and claim that the future belongs to Biophysics therole of which will be to determine precisely the spatial arrangement of glycans and theconformation of glycoproteins in order to understand the mechanism of their action. Themost exciting and marvellous age of the history of glycoproteins starts right now".

Ten years later, we can claim that the problem of determination of the primary structureof glycans is actually solved. The procedures have been miniaturized and applied to thestudy of cell membrane glycoproteins. In this field, the introduction of high-resolutionNMR has been a decisive event. In fact, this procedure is rapid, non-destructive andvery sensitive since it requires amounts of glycans varying from 25 to 100 micrograms only.

In the sane way, the recent intervention of the Biophysicistsleadto the definition of thespatial morphology of glycans and to the concept of their steric conformational changesfitting to the role they have to play. In fact, experimental results obtained in the past

two years by exploring the spatial conformation of glycans through physical methodsX-ray diffraction, nuclear magnetic resonance, electron paramagnetic resonance and small—angle neutron scattering, led to the fundamental finding that the antennae, which supportthe biologically active part of the glycans, are not rigid formations at all. On thecontrary, they must be considered as flexible and mobile parts of glycan molecules

876 J. MONTREUIL

solidly planted on the rigid arm constituted of the terminal trisaccharide linked to thepeptide chain. In fact, the trisaccharide arm probably fixes in part the conformation ofthe glycan, preventing the twist of the whole molecule, due to the relative rigidity of

the G1cNAc—Asn linkage (Ref. 86) . So could be explained why glycoasparagines are often morepotent inhibitors of haemagglutination by certain lectins : for example, the relativeamounts of glycan required to inhibit red cell agglutination by VLCAict ctbct agglutinin is8-fold higher than for the corresponding glycoasparagine (Ref. 58).

The concept of the antennae changing their conformation according to the environmentfits perfectly with the concept of glycans acting as recognition signals. Moreover, thedouble character of rigidity and flexibility of glycans may be central to the biologicalrole of the carbohydrate moiety of glycoproteins.

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