conglutinin with the complement glycopeptide iC3b rather than the intact glycoprotein C3 is due to the oligosaccharide accessibility rendered by proteolysis.
THEJOURNAL OF
BIOLOGICAL CHEMISTRY
Val. 264,No. 23,Issue of August 15,pp. 13834-13839,1989 Printed in U.S.A.
0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.
A Library of Oligosaccharide Probes (Neoglycolipids) from NGlycosylated Proteins RevealsThat Conglutinin Binds to Certain Complex-type as Well as High Mannose-type Oligosaccharide Chains* (Received for publication, March 28, 1989)
Tsuguo MizuochiSQ,Ruth W. LovelessSV, Alexander M. LawsonIJ,Wengang ChaiII, Peter J. Lachmann**,Robert A. ChildsS, Steffen ThielSS, and Ten FeiziS86 From the $Section of Glycoconjugate Research and the ))Section of Clinical Mass Spectrometry, Medical Research Council Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ, the **Molecular Immunopathology Unit, Medical Research Council Centre, University Medical School, Hills Road, Cambridge CB2 2QH, and the $$Medical Research Council Immunochemistry Unit, University of Oxford, South Parks Road, OxfordOX1 3QU, United Kingdom
This report describes the preparation of a library of oligosaccharide probes (neoglycolipids) from N-glycosylated proteins, characterization of the probes by liquid secondary ion mass spectrometry, and investigation of their reactions with ‘2SI-labeledbovine serum conglutinin by chromatogram binding assays. The results, together with additional binding studies using neoglycolipids derived from purified complex type bi-,tri-, and tetraantennary oligosaccharides from urine, or their glycosidase-treated products, have shown that the combining specificity of conglutinin includes structures not only on high mannose-type oligosaccharides but also on hybrid- and complex-type chains. With high mannose-type oligosaccharides there is increased reactivity from the Man5 tothe Mans structures, indicating a preference for the terminal Manal-2 sequence. With complex- and hybrid-type oligosaccharides, the requirements for binding are the presence of nonreducing terminal N-acetylglucosamine or mannose residues, but the presence of a bisecting N-acetylglucosamine residue may inhibit binding. From these results it is deduced that the reactivity of conglutinin with the complement glycopeptide iC3b rather than the intact glycoprotein C3 is due to the oligosaccharide accessibility rendered by proteolysis in thecomplement cascade.
In order to study the roles of glycoprotein oligosaccharides as recognition structures, a microtechnique has been under development (Tang et al., 1985; Stoll et al., 1988) which involves the conjugation of oligosaccharides or their alditols to the lipid phosphatidylethanolamine dipalmitoyl. The resulting neoglycolipids can then be resolved on thin layer chromatograms and used as solid-phase oligosaccharide probes as with natural glycosphingolipids (Magnani et al., 1980). The aim of the present study has been to generate a library of neoglycolipids that includes a range of oligosaccha-
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Supported by the Cancer Research Campaign. Present address: Division of Biomedical Polymer Science, Institute of Comprehensive Medical Science, Fujita-Gakuen Health University School of Medicine, Toyoake, Aichi 470-11, Japan. ll Supported by the Arthritis and Rheumatism Council. To whom correspondence and reprint requests should be addressed. Tel.: 01-864-5311. Fax: 01-423-1275.
ride structures from N-glycosylated proteins for use as ligands in studies of oligosaccharide recognition by, for example, the adhesins of infective agents(Rosenstein et al., 1988) and mammalian carbohydrate-bindingproteins(Childs et al., 1989) including bovine serum conglutinin, as presented here. Conglutinin binds in a calcium-dependent mannerto yeast polysaccharides and to erythrocytes that have been reacted with antibody and complement to generate the complement cleavage glycopeptide iC3b (Lachmann, 1967). We (Loveless et al., 1989) have recently shown that conglutinin is a lectin which binds certain nonreducing terminal N-acetylglucosamine, mannose, and fucose residues. Other studies (Hirani et al., 1985) have suggested that the natural ligand for conglutinin may be carried on N-linked oligosaccharides of high mannose-type on C ~ Cthe , major glycosylated fragment of iC3b, since treatment of this glycopeptide with endo-0-Nacetylglucosaminidase H abolished reactivity. Conglutinin is composed of 12 or more subunits of 48 kDa each containing two globular domainsseparated by a collagen-like region (Stranget al., 1986).A structural similarity has been suggested (Young and Leon, 1987) tothe calcium-dependent serum lectins known as mannose-binding proteins A and C on the basis of limited N-terminal sequence data (Davis and Lachmann, 1984). Thus, conglutinin may have been the first described member of the family of calcium-dependent endogenous lectins molecularly characterized by Drickamer (1988). We describe here (a) the generation of the neoglycolipids from oligosaccharides released by hydrazinolysis from several mammalian glycoproteins, or isolated from the urine of patients with GM,gangliosidosis, (6) structural identification of individual neoglycolipid bands by LSIMS’ directly from thin layer silica gel chromatograms, and (c) their reactivities in chromatogram binding assays with lZ5I-labeledconglutinin. MATERIALSANDMETHODS
Chemicals and Enzymes-Human and mouse IgG, human transferrin, bovine RNase B, hen ovalbumin, and phosphatidylethanolaminedipalmitoyl were purchased from Sigma Chemical CO. Ltd. (Poole, Dorset, United Kingdom). Aluminium backed high performance TLC plates, Silica Gel 60, were obtained from Merck Co. (Darmstadt, West Germany). Sialidase purified from Arthrobacter ureafaciens was purchased from NakaraiChemicals Ltd. (Kyoto, Japan); @-galactosidaseand @-N-acetylhexosaminidasepurified from jack bean meal were obtained from Seikagaku KogyoCo. (Tokyo, Japan). Oligosaccharides-Release of asparagine-linked oligosaccharides from glycoproteins by hydrazinolysis (Takasaki et al., 1982) and The abbreviation used is: LSIMS, liquid secondary ion mass spectrometry.
13834
Neoglycolipids from N-Glycosylated Proteins Bind Conglutinin
13835
enzymatic digestions of released oligosaccharides (Yamashita et al., and eluted with a 50-500 mM NaCl gradient. The conglutinin-con1982;Mizuochi et al., 1982)were performed as described previously. taining fractions were identified using an enzyme-linked absorbent Human and mouse IgG (Mizuochi et al., 1982; 1987; Harada et al., assay (Baatrup etal., 1987)and analyzed by sodium dodecyl sulfate1987)and human transferrin (Spik etal., 1975)are known to contain polyacrylamide gel electrophoresis using conditions as described by complex-type oligosaccharides; RNase B, high mannose-type oligo- Laemmli (1970). Chromatogram Binding Assays-The binding of 12sII-labeled conglusaccharides (Liang et al., 1980);and ovalbumin, both high mannosetype and hybrid-type oligosaccharides (Tai et al., 1977; Nomoto and tinin to neoglycolipids on silica gel chromatograms was assessed by Inoue, 1983).The oligosaccharide mixtures derived from human and autoradiography as described previously (Loveless et al., 1989). mouse IgG, and human transferrin were digested with A. ureafaciens sialidase to obtain asialooligosaccharide mixtures. Asialo, agalacto RESULTS oligosaccharide mixtures derived from human IgG and human transferrin were obtained by incubation of the respective asialooligosacStructural Identification by LSIMS of Neoglycolipids charidemixtureswithjackbean &galactosidase.Asialo-agalactofrom N-Glycosylated Proteins oligosaccharides lacking nonreducing terminal N-acetylglucosamine residues derived from human transferrin were obtained by sequential The positions of neoglycolipids derived from five glycoprodigestion of asialotransferrin oligosaccharides by jack bean @-galactosidase and jack bean @-N-acetylhexosaminidase.Oligosaccharides teins, separated by TLC and visualized by orcinol stain are from the urineof patients with Gwl gangliosidosis designated GMl-A, indicated in Fig. 1, lanes 1. These consisted of six bands (aGMl-B, and GM,-C, which, respectively, contain the structures Galal- f ) from human IgG, three (g-i) from mouse IgG, one (i) from 4GlcNAc~1-2Manal-3(Gal~1-4GlcNAc~l-2Manal-6)Man~l-4Glctransferrin, four (k-n) from RNase B, and multiple bands NAc, G a l ~ l - 4 G l c N A c ~ 1 - 2 ( G a l ~ l - 4 G l c N A c ~ l - 4 ) M a n n l - 3 ( G afrom l ~ l - ovalbumin. The molecular weights (Table I)of the main 4GlcNAc~l-2Manal-6)Man~l-4GlcNAc, andGalol-4GlcNAcBl-2components in bands a-n were derived from the intense (M (Gal~l-4GlcNAc~1-4)Mannl-3-[Gal~l-4GlcNAc~l-2(Gal~l4GlcNAc~l-6)Man~l-6]Man~l-4GlcNAc, weregiftsfromDr. A. - H)- ions produced by LSIMS. In addition, oligosaccharide sequence information was obtained from fragment ions in the Lundblad (Biocarb, Lund, Sweden). Preparation and Separationof Neoglycolipids-The neoglycolipids spectra of all but the three minor bands, d, f, and n. From derived fromasparagine-linked oligosaccharides of glycoproteins were these data, together with the knowledge of N-linked oligosacobtained by their conjugation to phosphatidylethanolamine dipalmi- charide structures of the corresponding glycoproteins, structoy1 as described by Stoll et al. (1988)with minor modifications as tural assignments could be made for the major components follows. Each oligosaccharide sample (25-50pg of carbohydrate) was mixed with 5 pl of water and 475 pg of phosphatidylethanolamine in bands a-n as follows. Molecular Weight Assignmentsand Sequence Informationdipalmitoyl dissolved in 95 pl of a mixture of chloroform/methanol, 1:l (v/v). After incubation at 60 "C for 2 h in a 1-ml reaction vial, LSIMS analysis of bands a, c, and e gave molecular ions (M 100 pg of sodium cyanoborohydride in 10 pl of methanol was added H)- 2137, 2299, and 2461 Da, respectively (Fig. 2), which and then thereaction was continued for 16 h a t 60 "C. The reaction correspond to the phosphatidylethanolamine dipalmitoyl conmixture containing 2.5 pg of oligosaccharide was subjected to TLC jugates of the agalactosyl, monogalactosyl, and digalactosyl using chloroform/methanol/water 105:10028 (byvolume). In this biantennary oligosaccharides, respectively, with core region TLC system, free oligosaccharides remain near the origin and the conjugates migrate faster. After development to approximately 7.5 fucose residues of human and mouse IgG (Table I; Mizuochi et al., 1982, 1987). Bands b, d, and f, which were detected in cm from origin, the TLC plateswere air-dried. Structural Assignments of the Neoglycolipids Separated on TLC the neoglycolipid mixture derived from human IgG but not Plates by LSZMS-For structural identification, the TLC/MS apmouse IgG, gave (M - H)-ions a t 2340,2502, and 2664 proach of KushiandHanda (1985) wasused. The neoglycolipid corresponding to the biantennary agalactosyl,mono-, and mixtures (-10 pg of carbohydrate/lane) were chromatographed. Areas corresponding to orcinol-stained bands visualized in parallel lanes digalactosyl, fucosyl oligosaccharides with bisectingN-acetylwere cut and subjected to LSIMS following addition of a matrix glucosamine residues (Table I). These areknown to occur in consisting of 3 pl of chloroform/methanol/water, 25258 (by volume) human, but notin mouse, IgG. LSIMS of band j from transand 3 pl of tetramethylurea/diethanolamine/meta-nitrobenzylalco- ferrin, which showed the samemobility on TLCas bands e/i, hol, 2:2:1 by volume). Negative ion mass spectra were obtained a t gave a molecular (M H)- ion a t 2315 Da which corresponds 1000 resolving power on a VG Analytical ZAB2-E mass spectrometer with an 11-2505data system using a cesium gun operated a t 35 keV A B C D E and emission current of 0.5 pA. In SituGlycosidase Treatment of the Neoglycolipids on TLCPlateAn area of the chromatogram containinga mixture of neoglycolipids derived from asialo human I g G oligosaccharides was cut, dipped for 1 min in 0.05% (w/v) solution of Plexigum P28 (Cornelius Chemical Co. Ltd., Romford, U.K.) in n-hexane and immersed for 2 h in 3% bovine serum albumin/phosphate-buffered saline (20 mM sodium mM NaCI, 0.05% NaN3) solution.After phosphate buffer, pH 7.4, 150 rinsing with 0.1 M citrate phosphatebuffer, pH 3.5,the chromatogram piece was overlaid with a solution of @galactosidase, 3.25 units/ml in 0.1 M citrate phosphate buffer, pH 3.5 (or with the buffer alone as a control), and incubateda t 37 "C for 17 h. Conglutinin-The conglutinin was isolated from heat-inactivated 1 2 1 2 1 2 1 2 1 2 bovine serum by two different procedures. Procedure A was by abFIG.1. Silica gelchromatography of neoglycolipids derived sorptionandelutionfromyeast cell walls (Lachmann, 1962). In procedure B bovine serum was dialyzed extensivelyagainst deionized from glycoprotein oligosaccharides, and chromatogram bindconglutinin. Neoglycolipids (2.5pg water a t 4 "C and centrifugeda t 10,000X g for 20 min. The precipitate ing assays with 1Z61-labeled dissolved in Tris-buffered saline/Ca*' (10 mM Tris/HCl buffer, pH of carbohydrate/lane) derived from asialo oligosaccharides of human 7.4, 150 mM NaCI, 0.05% NaN3, 0.05 Tween 20, containing 10 mM IgG (A), mouse IgG ( B ) and transferrin (C), and from RNase B (D) CaCI2) was applied to a mannan-absorbentcolumnprepared by and ovalbumin( E ) were chromatographed and theirreactivities with conjugating yeast mannan (Nakajima andBallou, 1974)to Sepharose conglutinin assessed by autoradiography (lanes 2, whitebands) as (Pharmacia LKBBiotechnology Inc.) activated by cyanogen bromide described under "Materials andMethods." The same laneswere then (March etal., 1974);the column was washed in Tris-buffered saline/ stained with orcinol reagent (lanes I , dark bands) to visualize the Ca*' and eluted with Tris-buffered saline containing 10 mM EDTA. neoglycolipid bands designated a-n; bands 6, d, and f were faintly stained. Autoradiographywas for24 h. Block arrows indicatepositions The eluted conglutinin was further purified by ion exchange chromatography using a Mono Q (Pharmacia) column equilibrated in 10 of sample application; white arrows indicate bands faintly reactive mM Tris/HCI buffer, pH 7.8,50mM NaCI, 1 mM EDTA, 0.05% NaN3 with conglutinin.
-
-
Neoglycolipids from N-Glycosylated Proteins Conglutinin Bind
13836
TABLEI Structures of neoglycolipids identified by LSIMS analysis on silica gel chromatograms Bands
structures+
:" a,g
2137.1
+
6
GNBl-ZMal,
91-4GNBl-4GN-PPEADP GNB1-2Mol
GNB
GNBl-2Mak b
2340.1
Fa
1 ' t, $B1-4GNB1-4GN-PPEADP
GNB1-2Ma1' Fa
6
GB1-4GNB1-2Ma1, 2299.1
6/3MBl-4GNB1-4GN-PPEADP GNB1-2Mm1P16
GNB
t
GB1-4GNB1-ZM.1, d
Fa I
~$$B1-4GNB1-4~N-PPEADP
2502.2 CNB1-2M.1'
:"
t
GB1-4GNB1-2Mm1,
63MB1-4GNB1-4GN-PPEADP
2461.2 GB1-4GNBl-ZMal'
GNB
GB1-4GNBl-2Mn1\ 2664.2
Fa 1 4 6MBl-4GN51-4GN-PPEADP
b
GB1-4GNB1-2Mol2
CB1-4GNB1-2Mal\ 2315.1
1909 .O
:WB1-4GNBl"&N-PPEADP GB1-4GNB1-2Ma1°
[ ""
;Mal,
1
:HB1-4GNB1-4GN-PPEADP Mal'
2071.0
m
2233.1
n
2395.1
*Calculated theoretical values for (M-H)- ions. These were confirmed from LSIMS spectra, although it should be noted that the intensity of the [(M-H)+l]-ieotape peak in the i o n clueter exceeds that of the manoisotopic M-Hf-ion. +, sequence information obtained from fragment i o n s . +F, fucose; G , galactose; CN, K-aeetylglucosamine; M, mannose.
r
main series of fragment ions present,' only those arising by @-cleavageof glycosidic bonds with hydrogen rearrangement have been numbered in the figures. Secondary fragmentation ions were not observed, and hence branching could be detected. In Fig. 3, for example, the branching pattern of the structure in band k is uniquely defined by the ions m/z 1746, 1422, and 1098 from losses of 1, 3, and 5 mannose units, respectively, while m/z 895 indicates the lack of a fucose on the N-acetylglucosamine residue linked to phosphatidylethanolamine dipalmitoyl. Core region fucose residues in neoglycolipid bands a, c, and e are evident first, from ions m/z 1991, 2153, and 2315 (Fig. 2, A, B, and C, respectively) which arise from loss of fucose by P-cleavage with hydrogen rearrangement (M - H - 146)- and second, from the occurrence of the ion m/z 1041. In addition, the biantennary pattern of each could be deduced from the composition indicated by the (M - H)- ion and the presence of the specific fragment ions shown in Fig. 2. Sequence information was similarly obtained for the main components of neoglycolipid bands b, g-j, 1, and m. By this means, the oligosaccharides characteristic of transferrin and RNase B were all identified, and among the 16 oligosaccharides known to occur in human IgG the 8 major fuco-oligosaccharides accounting for 85% of total were identified as phosphatidylethanolamine dipalmitoyl conjugates; the corresponding afucosyl analogues (15% of IgG oligosaccharides) could not be distinguished by LSIMS from their comigrating fucosyl analogues with the amountsanalyzed. Partial Dehydration at Reducing End N-Acetylglucosamine-Derivatization of these glycoprotein-derived oligosaccharides, all of which have N-acetylglucosamine at thereducing end, was accompanied by a partial dehydration of the linkage sugar giving (M - H - 18)-, as shown by the ions with asterisks in Fig. 2. This artefact was also observed with the GMl gangliosidosis oligosaccharides (results not shown) and with oligomers derived from chitin.' Conditions for minimizing this dehydration or carrying it to completion are under investigation. Reactivities with Conglutinin Binding Studies with the Library of Oligosaccharide Probes Derived from Glycoproteins-In chromatogram binding assays, lz5I-1abeledconglutinin purified by both procedures A and B gave the same results. There was selective binding to bands a and c derived from human IgG, bands g and h from mouse IgG, and bandsk-n from RNase B, andseveral bands from ovalbumin (Fig. 1, A-E, lanes 2 ) . These results indicated that among biantennary complex type oligosaccharides, those with nonreducing terminal N-acetylglucosamine residues are recognized by conglutinin, but only in the absence of the bisecting N-acetylglucosamine residues. The digalactosyl oligosaccharides are not recognized. With the high mannosetype oligosaccharides, the increasing reactivity from the Man5 to the Mans structures indicates that conglutinin can react with the oligosaccharides that have nonreducing terminal Elanal-3 and Manal-6 sequences, but has a preference for oligosaccharides terminating with theManal-2 sequence. With the mixture of neoglycolipids derived from ovalbumin oligosaccharides, multiple components were bound by conglutinin; this is consistent with previous knowledge that the oligosaccharides of this glycoprotein are a heterogeneous mixture of hybrid-type (some with terminal N-acetylglucosamine
to the phosphatidylethanolamine dipalmitoyl conjugate of the same biantennary structure ase/i but lacking the core region fucose residue (Table I; Spik et al., 1975). Examples of sequence-determining fragment ions obtained by LSIMS which permitted structural assignment are shown A. M. Lawson, W. Chai, G . Cashmore, M. S. Stoll, E. F. Hounsell, in Fig. 2 for bands a, c, and e and in Fig. 3 for band k. Of four and T.Feizi, Carbohydr. Res., submitted for publication.
13837
Neoglycolipids from N-Glycosyluted Proteins Bind Conglutinin
e
2137
1244
!934177, I
I-
I
I_
1244
Gal+GlcNAc+Man I 1-+""-I h~an/GlcNAc+GlcNAc-P GlcNAciMan? 1041 'iOS61934
1934
C
1041
2299
2299.1
2153
F~~ I-
2137
1'
b
b
2153
2 4 6 1.2
?OS6 1934
n
ap
-88 %
c .In
c 68 Q)
c
- 48 C Q)
.-e>
228 Q) a e lie
2288
mlz
FIG. 2. Analysis by LSIMS of three neoglycolipid bands a, c, and e derived from human IgG. Neoglycolipids derived from the asialo oligosaccharides of human IgG were resolved on silica gel chromatograms, areas corresponding to orcinol-stained bands were cut andsubjected to LSIMS as described under "Materials and Methods." The results shown are for band a ( A ) ,band c ( B ) , and band e ( C ) as defined in Fig. 1. Molecular ions (M - H)- and fragment ions arising from 0-cleavage of glycosidic bonds, rounded down to integer values, are indicated. Asterisks indicate ions (M - H - 18)-,produced by partial dehydration a t the N-acetylglucosamine residues linked to lipid. P = phosphatidylethanolamine dipalmitoyl.
residues) and high mannose-type (Tai et al., 1977; Nomoto and Inoue, 1983). Recognition of biantennary oligosaccharides terminating with N-acetylglucosamine was confirmed by binding studies with neoglycolipids derived from @-galactosidase-treated asialooligosaccharides from human IgG (Fig. 4-4). The major orcinol-stained derivative thus obtained (Fig. 4 A , lune 3 ) chromatographed in the same position as band a (lune I) and reacted strongly with '251-labeled conglutinin. In a separate
experiment where the neoglycolipids from asialooligosaccharides of human IgG were treated with @-galactosidasein situ after TLC,a third band reactive with conglutinincorresponding to agalacto band e was revealed (Fig. 4B,lune 2). Additional conglutinin binding experiments were performed with neoglycolipids derived from asialooligosaccharides of transferrin that had been further digested with @galactosidase followedby @-N-acetylhexosaminidasebefore conjugation. The main orcinol-stained band obtainedafter @-
Neoglycolipids from N-Glycosyluted Proteins Bind
13838
FIG. 3. Analysis by LSIMS of the neoglycolipid band k derived from RNase B. Neoglycolipids derived from the oligosaccharides of RNase B were resolved on a silica gel chromatogram, areas corresponding to orcinol-stained bands were cut and subjected to LSIMS as described under "Materials and Methods." The results shown are for band k. Positions of the molecular ion (M - H)and of fragment ions arising from 8cleavage of glycosidic bonds are indicated. Abbreviations are as defined in Fig. 2.
A
1
1
4
1909.0
1746 A
E On 2
'E
68
2
5 (n
$ 28
3 a
B
3
Conglutinin
C
1 2
3
4
n
5
6
1 2
3 4
5
6
7
891011121314
FIG.4. Unmasking of recognition structures for conglutinin on complex-type oligosaccharides following glycosidase treatments of free oligosaccharides (before conjugation to lipid) or of neoglycolipids on chromatograms. A, lanes 2 and 4, shows the binding of "'I-labeled conglutinin detected by autoradiography to neoglycolipids derived from asialo human I g G oligosaccharides before (lane 2) and after (lane 4 ) digestion with 8-galactosidase; lanes 1 and 3, respectively, show the same lanes stainedwith orcinol reagent after autoradiography. B shows the binding of 1251-labeledconglutinin detected by autoradiography to neoglycolipids derived from asialo human IgG oligosaccharides which were digested with @-galactosidasein situ after TLC (lane 2); the same lane (now designated lane I ) was then stained with orcinol reagent. C shows the lack of binding of 1251-labeledconglutinin to neoglycolipids derived from asialotransferrin oligosaccharides (lane 2 ) and binding to neoglycolipids derived from asialotransferrin oligosaccharides that had been digested with @-galactosidase(lane 4 ) and further digested with 8-N-acetylhexosaminidase(lane 6). Lanes 1,3, and5 represent the same lanes stained with orcinol reagent after autoradiography. D shows lack of binding of 1251-labeledconglutinin to the neoglycolipids derived from the galactose-terminating biantennary ( h n e 2), triantennary (lane 8), and tetraantennary (lane 12) oligosaccharides from G Mgangliosidosis ~ urine (for structures see "Materials and Methods"); this contrasts with binding to the neoglycolipids derived from the 8-galactosidase-digested oligosaccharides (lanes 4, 10, and 14, respectively) and from the biantennary oligosaccharides which had been further digested with 8-N-acetylglucosaminidase (lane 6). After autoradiography the same lanes were stained with orcinol reagent; these are shown as the odd-numbered lanes adjacent to the autoradiography lanes. Bands a-f in A and B , lanes 1 , and thepurpose of the white and black arrows correspond to those in Fig. 1. -2.5 pg of carbohydrate was applied per lane.
galactosidase treatment reacted strongly with conglutinin (Fig. 4C, lane 4). Since the biantennary oligosaccharides of transferrin differ from IgG oligosaccharides in lacking a core region fucose residue (Spik et al., 1975; Mizuochiet al., 1982, 1987), the results indicate that there is no requirement for a core region fucoseresidue for reaction with conglutinin. Binding to theproduct after @-N-acetylhexosaminidase treatment (Fig. 4C,lane 6 ) was less intense relative to the @-galactosidase-treated product, indicating that conglutinin can react, albeit weakly, with the trimannosyl core. Binding Studies with Probes Derived from Purified Oligosaccharides-Chromatogram binding experiments were also performed with neoglycolipids derived from the purified bi-, tri-, and tetraantennary oligosaccharides from urine, GMI-A, GMl-B and GM,-C,all of which have a single N-acetylglucosamine at the reducing end. In each case conglutinin binding was observed with the @-galactosidase-treatedproducts (Fig. 40, lanes 4,10, and 14) but notthe untreated oligosaccharides (lunes2,8, and 12). As with the biantennary oligosaccharides from transferrin, the neoglycolipid derived from Gw1-A after sequential treatments with @-galactosidaseand @-N-acetylhexosaminidase, was bound weakly by conglutinin (lane 6). These results show that the intact chitobiosyl core is not required for reactivity of N-linked chains with conglutinin.
DISCUSSION
The special features of this investigation are (a) the generation of a library of oligosaccharide probes in the form of phosphatidylethanolamine dipalmitoyl conjugates, encompassing a spectrum of complex-type and high mannose-type oligosaccharides from N-glycosylated proteins, (b) the demonstration of the excellent ionization characteristics of the conjugates such that themain components could beidentified from the molecular weightand sequence information obtained by LSIMS analyses directly on silica gel chromatograms, and (c) the successful use of mixtures of glycoprotein-derived oligosaccharide probes in conjunction with specific glycosidases in studies of the glycoprotein oligosaccharides recognized by conglutinin. The probes generated and characterized in this study have been used in other studies involving the mannose-binding proteins of human and rat serum (Childs et al., 1989) and type I fimbriated Escherichia coli (Rosenstein et al., 1988). The results of the conglutinin-binding experiments indicate that complex-type chains with unsubstituted terminal Nacetylglucosamine residues on their outer branches and unsubstituted mannose residues in their core regions are recognized by this protein, as well as theterminal mannose residues
Neoglycolipids from N-Glycosylated Proteins Bind Conglutinin
13839
* Fuca
1
I
4
*
*
Gal~l-3GlcNAc~l-3Gal~l-4GlcNAc~l-ZManal ',
*
6
Manp1-4GlcNAcpl-4GlcNAc
3 / G a l p l - 4*G l c N A c p l - 2 M a n a l
*
of high mannose-type chains. The results with IgG oligosaccharides suggest that on the complex type chains a bisecting N-acetylglucosamine residue inhibits binding. It will be necessary to confirm this using purified oligosaccharides. Neither the core region fucose residue nor the reducing end N-acetylglucosamine residue are recognized by this protein. From these results, together withthe earlier observations (Loveless et al., 1989) that conglutinin binds to simple sugars terminating in the sequences GlcNAcP1-4, Manal-3,andFucal4(GalPl-3)GlcNAc, it is predicted that this protein has the potential to bind to an array of glycoconjugates, and in particular to those that containN-linked oligosaccharides of high mannose, hybrid, and complex types. The latter may have outer chains terminating with N-acetylglucosamine or they may have extended outer chains fucosylated at N-acetylglucosamine as indicated above, where asterisks (*) indicate residues predicted to be recognized by conglutinin if present as nonreducing terminal structures. The lack of reactivity with the native glycoproteins C3 and IgG which contain such recognition structures indicates that their protein moieties hinder the accessibility of the oligosaccharides for reaction with conglutinin. Enhanced oligosaccharide accessibility is therefore likely to be the biochemical basis for the binding of conglutinin to the proteolytically generated and cell-bound iC3b fragment of the human complement component C3 on which high mannose-type oligosaccharides have been identified (Hase et al., 1985; Hirani et al., 1986). The oligosaccharide binding specificity of conglutinin has an overall similarity to those of the soluble mannose-binding proteins of the rat and man that have been similarly investigated (Childs et al., 1989), but there are subtle differences in their reaction patterns with the oligosaccharide probes; for example, the mannose-binding proteinsclearly react with the biantennary oligosaccharides with bisecting N-acetylglucosamine. Another difference is that the mannose-binding proteins of human and ratserum (Kawasakiet al., 1985) and the recombinant carbohydraterecognition domain of the ratmannose-binding protein3 do not bind toerythrocytes coated with iC3b. The molecular basis of this difference requires further investigation. Wewould predict that the residual protein moiety on iC3b influences the conformation or accessibility of the oligosaccharides in such away that theycan be accommodated by the combining site of conglutinin but notby those of the mannose-binding proteins. Clearly, the potential is R. W. Loveless, K. Drickamer, and T. Feizi, unpublished observations.
there for conglutinin, as with the mannose-binding proteins, to react with glycoconjugates other than the products of the complement component C3, but a major determining factor is likely to be the accessibility of oligosaccharides to their respective carbohydrate-binding sites. Acknowledgment-We are grateful to Maureen Moriarty for the preparation of the manuscript. Addendum-Partial protein sequencing of bovine conglutinin has revealed that it contains some of the highly conserved residues of calcium-dependent carbohydrate recognition domains (S. T h i e l and T. Willis, unpublished observations). REFERENCES Baatrup, G., Thiel, S., Isager, H., Svehag, %-E., and Jensenius, J. C. (1987) S c a d J. Immunol. 26,355-361 Childs, R. A., Drickamer, K., Kawasaki, T., Thiel, S., Mizuochi, T., and Feizi, T. (1989) Biochem. J., in press Davis, A. E., 111, and Lachmann, P.J. (1984) Biochemistry 23,2139-2144 Drickamer, K. (1988) J. Btol. Chern. 263,9557-9560 Harada, H., Kamei, M., Tokumoto, Y., Yui, S., Koyama, F., Kochibe, N., Edno, T., and Kohata, A. (1987) Anal. Btochem. 164,374-381 Hase, S., Kikuchi, N., Ikenaka, T., and Inoue, K. (1985) J. Biochem. (Tokyo)
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