0161-5890/92 $5.00 + 0.00 Pergamon Press Ltd

Molecular Immunology, Vol. 29, No. 12, pp. 1437-1446, 1992 Printed in Great Britain.

LOCALIZATION AND CHARACTERIZATION OF THE CARBOHYDRATE-BINDING SITE OF THE PORCINE LYMPHOCYTE MANNAN-BINDING PROTEIN* KAREL BEZOUSKA,~ VLADIMIR E. PISKAREV,~ GOVERT J. VAN DAM,@MILOSLAV POSP%IL,117 JAROSLAVKUBRYCHT~(and JAN KOCOUREK** tInstitute of Biotechnology and **Department of Biochemistry, Faculty of Science, Charles University Prague, Albertov 2030, CS-12840 Praha 2, Czechoslovakia; SInstitute of Organo-Metallic Compounds, Russian Academy of Sciences, 117813 Moscow, Russian Federation; GLaboratory of Parasitology, Institute of Tropical Medicine, Medical Faculty, University of Leiden, The Netherlands; and //Department of Immunology, Institute of Microbiology, Czechoslovak Academy of Sciences, Videfiska 1083, CS-14220 Praha 4, Czechoslovakia (First received 10 March 1992; accepted in revised form 8 July 1992) Abstract-Mannan-binding proteins found in the liver and serum of several vertebrate species are supposed to play an important role in the intracellular transport of glycoproteins, as well as in several protective reactions including complement activation and elimination of various pathogens. To study these protective functions at molecular level it is necessary to understand the fine oligosaccharide specificity and mutual relation among various forms of these soluble lectins. We have isolated mannan-binding protein as peripheral membrane proteins of porcine lymphocytes. This lectin was purified to homogeneity and shown to possess many properties in common with the well studied rat liver proteins (mol. mass, subunit composition and general organization of the molecule). Binding studies performed with three series of defined oligosaccharides (high mannose, hybrid type, and complex) on native lectin molecules as well as isolated carbohydrate-binding domains revealed distinctive features of this mannan-binding protein, including its impaired ability to bind the oligosaccharide ligand after reduction and decyclization at core N-acetyl-o-glucosamine 1.

INTRODUCTION Mannan-binding proteins (MBPs) have been isolated from liver (Kawasaki et al., 1978) and serum (Kawasaki et al., 1983; Taylor and Summerfield, 1984; Summerfield and Taylor, 1986) of several vertebrate species by affinity chromatography on immobilized mannoproteins and characterized as multimeric proteins composed of subunits of approximately 30 kDa. The unusual kinetics of secretion of these proteins has been observed in pulse labelling experiments (Brownell et al., 1984) but it was not before the methods of molecular genetics had been applied to their study that the general structure of the molecule has become apparent (Drickamer et al., 1986; Drickamer and McCreary, 1987). The molecule is composed of a short N-terminal peptide rich in cysteine and responsible for the formation of multimers by disulfide bonds, followed by a collagen domain constituted of 18-20 repeats of the sequence Gly-X-Y and the C-terminal domain bearing the car-

*This is paper number LXXX1 of the series “Studies on Lectins”. TAuthor to whom correspondence should be addressed. Abbreviations: MBP, mannan-binding protein; PEG, polyethylene glycol; BSA, bovine serum albumin; RCA, Ricinus communis agglutinin; SBA, soybean agglutinin; EDTA, ethylenediamine tetra-acetate.

bohydrate-binding site. This carbohydrate-recognition domain has significant homology with other glycoprotein receptors (Drickamer, 1988) and can be prepared by collagenase digestion (Drickamer et al., 1986). Because of its structural organization this lectin must undergo quite complicated post-translational modifications (Colley and Baenziger, 1987) and is considered to be a member of a more extended group of animal lectins having the collagen-like sequences (Thiel and Reid, 1989). This assumption has been supported by supercomputer-assisted construction of the evolutionary tree of the C-type lectin family (BezouSka et al., 1991) that includes receptors for endocytosis of partially deglycosylated glycoproteins (Ashwell and Harford, 1982), immunoglobulin receptors of lymphocytes (Ikuta et al., 1987), specific activating receptors of natural killer cells (Giorda et al., 1991), non-immune protective proteins (Ezekowitz, 1991) and cell adhesion and homing molecules of leukocytes-selectins (Lasky et al., 1989; Johnston et al., 1989; Bevilacqua et al., 1989). In the above evolutionary tree, mannose-binding proteins form group III of lectins together with conglutinin (Lee et al., 1991a) and pulmonary surfactant apoproteins (Benson et al., 1985). Another molecule within the C-type lectin family that binds specifically mannosecontaining glycoconjugate is the mannose receptor of macrophages and liver endothelial cells (Ezekowitz and Stahl, 1988; Lennartz et al., 1987). The structure of the 1437

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K. BEZOUSKA et al.

mannose receptor, however, is entirely different from that of the soluble mannose-binding proteins (Taylor et al., 1990): it contains eight carbohydrate recognition domains characteristic for the C-type lectin family within a single polypeptide and its position in the evolutionary tree indicates the evolutionary and functional relation to the other type II endocytic receptors (Kim et al., 1992). Mannose-binding proteins may be physiologically important in the transport of secreted glycoproteins between various intracellular compartments (Newton et al., 1987) and certain serum glycoproteins have been recently identified as their possible endogenous ligands (Mori et al., 1988). Moreover, these lectins are probably involved in protective reactions against various microorganisms coated with mannose-rich oligosaccharides (Ezekowitz, 1991). They are typical acute phase proteins (Ezekowitz et al., 1988; Taylor et al., 1989) that can activate complement by the classical pathway (Ikeda et al., 1987; Ohta et al., 1990) and mediate opsonization of various pathogens (Kuhlman et al., 1989) and protection against viruses (Ezekowitz et al., 1989). The important protective role of mannose-binding proteins has been supported by recent clinical studies relating the deficiency of these proteins to the impairment of certain reactions of non-specific immunity (Super et al., 1989; Sumyia et al., 1991). Mannose lectins recognize the core and peptide region of glycopeptide ligands (Colley et al., 1988) and this is why the term ‘core-specific lectin’ has been coined for these molecules (Maynard and Baenziger, 1982). More detailed investigation of the fine carbohydrate specificity indicates important differences between two different forms of the recombinant rat mannose-binding protein at the level of oligosaccharide binding (Childs et al., 1990). Moreover, additional molecular details of the recognition of carbohydrates by the lectin domains of these proteins have been revealed recently by inhibition studies (Lee et al., 1991b), X-ray analysis of crystals (Weis et al., 1991) and random mutagenesis studies (Quesenberry and Drickamer, 1992). In our laboratory we have developed new oligosaccharide derivatives retaining the native conformation of glycoprotein glycans (Piskarev et al., 19906) and evaluated the usefulness of these compounds for the structural investigation of the binding sites of plant (Piskarev et al., 1990a) and animal (BezouSka et al., 1985) lectins. Also, we have recently isolated new mannose-specific lectins from porcine lymphocytes (BezouSka et al., 1990). Thus, it seemed interesting to compare the specificity of these lectins with those from other species. Because of the ‘core’ specificity of the other described mammalian mannose-binding proteins, we wanted to test the influence of the structural alteration in this region of the oligosaccharide after reduction and decyclization of the terminal core N-acetyl-D-glucosamine 1. In this study we report the fine oligosaccharide specificity of porcine lymphocyte mannan-binding protein and its collagenase fragment for such oligosaccharide ligands.

MATERIALS

AND METHODS

Materials HPLC acetonitrile LiChrosolv, PEG 6000 and silica HPTLC plates were from Merck, Darmstadt, F.R.G.; 3-(4-hydroxyphenyl) propionic acid N-hydroxysuccinimide ester (Bolton-Hunter reagent) and chemicals for the preparation of polyacrylamide gels were from Serva, Heidelberg, F.R.G.; Ficoll 400, Sephadex G-15, Sephadex G-25, Sephacryl S-300 Superfine, CNBr-activated Sepharose 4B and protein mol. wt standards were from Pharmacia-LKB Biotechnology, Uppsala, Sweden; BSA (20% solution) from Sevac, Praha, Czechoslovakia; porcine pepsine and collagenase from C. histolyticum was from Sigma, St. Louis, MO, U.S.A. Yeast mannan was prepared from bakers’ yeast (S. cerevisiae) according to Nakajima and Ballou (1974) and coupled to CNBr-activated Sepharose 4B according to instructions from the manufacturer. Glycopeptidase A was prepared from almond meal (Taga et al., 1984) and b-hexosaminidase from jack bean meal (Li and Li, 1972). All other chemicals were analytical grade reagents. Bu&rs Buffer A: 0.02 M Tris-HCl, 0.15 M NaCl, CaCl,, pH 7.8; Buffer B: 0.02 M Tris-HCl, NaCl, 10mM EDTA, pH 7.8. Oligosaccharides

(numbering

and structures

10 mM 0.15 M

in Table 1)

Numbering of oligosaccharides was according to Tomiya et al. (1988). Oligosaccharides used in this study were prepared from glycoproteins indicated in Table 1 by the following procedure: glycoproteins were digested by porcine pepsin and glycopeptidase A (Tomiya et al., 1988) or reductive cleavage (Likhosherstov et al., 1988) and the released oligosaccharides purified by gel filtration and ion exchange chromatography on Dowex 50W column. Oligosaccharides eluted by distilled water were reduced with NaBH, or converted into /3-glycosylamines and derivatized with Bolton-Hunter reagent (Piskarev et al., 1988). Oligosaccharide mixtures were then separated by gel filtration on BioGel P4 column and HPLC on amino silica and ODS silica packings (BezouSka et al., 1990). Quantitative carbohydrate analysis (Porter, 1975) and proton nuclear magnetic resonance spectroscopy (Vliegenthart et al., 1983) were employed to confirm the anticipated type of structure. The preparation, isolation to homogeneity and methods of structural analysis for oligosaccharides M 6.1, M 7.1 and M 8.1 from legume storage proteins (Neeser et al., 1985), oligosaccharide 200.1 from IgG (Piskarev et al., 1990a), oligosaccharides 201 .l and 301.1 from egg white riboflavin-binding globulin (Likhosherstov et al., 1987) and oligosaccharide 501.1 from egg white ovomucoid (Piskarev et al., 1990b) have been described previously. Oligosaccharides from ovalbumin were fractionated as glycoasparagines after Pronase digestion (Huang et al., 1970), oligosaccharides released by hydrazinolysis (Takasaki et al., 1982), converted into 4-hydroxyphenylpropionyl derivatives (Piskarev et al.,

Characterization

of porcine mannan-binding

J

d t

protein

1439

K. BEZOUSKAet al.

1440

a ._ 8 g %

cd t

d

T

t

8 m

d t

d t

1442

K.

BEZOUSKA

1988) and after the final purification by HPLC oligosaccharides M 4.1, M 5.1, H 5.4, H 5.3, H 5.1 and H 4.3 were analysed by 500 MHz ‘H NMR spectroscopy (Ceccarini et al., 1984). Oligosaccharide M 3.1 was prepared from oligosaccharide 200.1 by enzymatic digestion with /J-hexosaminidase (Li and Li, 1970), separation of products by HPLC and characterized by compositional analysis and ‘H NMR (Piskarev et al., 1990~). Oligosaccharides from orosomucoid were separated after enzymatic release and reaction with the BoltonHunter reagent (Piskarev et al., 1988) on a semipreparative HPLC column (Separon SGX-NH,, 7 pm, 8 x 250mm, Tessek, Praha, Czechoslovakia) in acetonitrile-phosphate buffer gradients. The column was equilibrated at 2 ml/min with the initial solvent A (acetonitrile: 15 mM KH,PO, pH 5.2, 65: 35) and after the application of sample, eluted for 20 min with the solvent A and then a linear gradient to solvent B (acetonitrile : 15 mM KH,PO, pH 5.2, 50 : 50) in 120 min. Five major and several minor peaks were detected at 280 nm. The second and fourth major peak containing the complex type triantennary and tetraantennary oligosaccharides respectively, were treated with fl-hexosaminidase (Li and Li, 1970) repurified by HPLC using the same gradient system and the major peak for each repurification containing the oligosaccharide 300.1 and 400.1 were desalted on Sephadex G-15 and their structure analysed by ‘H NMR as described previously (Vliegenthart et al., 1983). Isolation of lymphocytes

mannan -binding

proteins

from

porcine

Porcine lymphocytes were prepared from heparinized pig peripheral blood by Ficoll-Verografin gradient centrifugation (PospiSil et al., 1986) and extracted for 30 min at 4°C in buffer B. The released peripheral proteins were separated from cells by centrifugation at 500g for 10 min, supplemented with Ca2+ to the final concn 10 mM, centrifuged again at 100,OOOg for 30 min and the clear supernatant applied to an affinity column (3 x 10 cm) with immobilized yeast mannan. The column was washed with 10 volumes of buffer A and mannan-binding proteins were eluted with buffer B. Eluted protein was dialysed against buffer A and concentrated by ultrafiltration. This preparation was further purified by gel filtration on Sephacryl S-300 (1 x 100 cm column) eluted with buffer A and affinity chromatography (Drickamer et al., 1986). Preparation

of carbohydrate-recognition

domain

The native lectin molecule was digested by collagenase (Drickamer et al., 1986) and the collagen-resistant fragment separated by gel filtration on Sephacryl S-300 and affinity chromatography. The purity of the native lectin and its carbohydrate-binding domain was checked by SDS electrophoresis (Laemmli, 1970). Binding studies Inhibition experiments were performed with reduced or BH-modified oligosaccharides and constant amount

rt al.

of ‘251-labelled oligosaccharide M 3.1-BH as described previously (Piskarev et al., 1990a). Apparent association constants of oligosaccharides for the multimeric and monomeric form of the lectin were determined by the PEG precipitation assay as modified by Grant and Kaderbhai (1986) as well as by several equilibrium techniques, which included gel filtration (Hummel and Dreyer, 1962) equilibrium dialysis performed in a multiple-chamber thermostated apparatus or Eppendorf microtubes (Reinard and Jacobsen, 1989) and frontal affinity chromatography (Ohyama et al., 1985). Results of binding experiments were evaluated according to Scatchard (1949).

RESULTS AND DISCUSSION

Although the specificity of several vertebrate mannanbinding proteins for carbohydrates and glycopeptides has been reported, for the investigation of fine oligosaccharide specificity of these lectins it was necessary to prepare series of well-defined oligosaccharides derived from N-linked glycoprotein glycans. Because of the core-region specificity of mannose lectins it was desirable to prepare structurally defined compounds that would still retain the native conformation of the core N-acetylD-glucosamine 1 and the adjacent N-glycosidic linkage to asparagine. A new derivatization procedure published recently by Piskarev et al. (1988) seems to be ideally suited for these requirements: when derivatized with Bolton-Hunter reagent the oligosaccharides prepared according to this modification scheme have essentially the same structure as found in natural glycoproteins. Moreover, the 4-hydroxyphenyl group replacing the asparagine is suitable for radioiodination and facilitates the detection of these compounds. Employing the above discussed procedure we have succeeded in preparation of three series of oligosaccharides in quantities sufficient for their structural analysis (Table 1). These series include high mannose, hybrid and complex oligosaccharides from various freely available glycoproteins. Two reduced oligosaccharides have been included to study the effect of reduction (and decyclisation) on the interaction with the lectin. Porcine lymphocyte mannan-binding protein has been isolated by affinity chromatography as the major purification step and its native molecular mass estimated to be approximately 190 kDa by gel filtration (Fig. 1). This indicates that, similarly to other mannan-binding proteins in vertebrates the native molecule exists as a hexamer, as the molecular mass of the subunit has been estimated to be about 30 kDa under reducing conditions (Fig. 2). The collagenase-resistant fragment of the native molecule has a molecular mass of 20 kDa on gel filtration as well as SDSSelectrophoresis and its isolation by affinity chromatography indicates the presence of the binding site for carbohydrate. Thus, the general organization of the molecule of porcine lymphocyte MBP is very similar to that reported for MBPs from other mammalian species.

Characterization

0.2

8

2 0 I

of porcine mannan-binding

. Ribanuclease C Fragment \ l Chymotrypsinogen A \

0.2

(8) Fragment

(Al Native complex

t-/L

\

0

%

a

I

I 40

I 80

.BSA

01

\ 1

I

0

1443

protein

I 120

I

0

I 40

I 120

I

80

Elution volume (ml)

Elution volume (ml)

Fig. 1. Analysis of porcine lymphocyte mannan-binding eluted by buffer A ~thout Table 2 summarizes

the results of binding experiments performed under non-equilibrium (PEG precipitation assay) and equilibrium conditions (other methods). From the data presented here several conclusions concerning the binding specificity of porcine lymphocyte mannan-binding protein can be deduced. First, the ‘core specificity’ of this lectin could be confirmed by the fact that the trimannosyl core oligosaccharide M 3.1 is the best ligand for the lectin while its substitution by the additional mannoses (M 4.1-M 8.1) or N-acetyl-oglucosamines (200.1-501.1) causes in all instances the decrease in affinity. Second, the strict requirement for the native confo~ation of the core oligosaccharide is indicated by experiments with reduced oligosaccha~des (M 3.101 and M 8.101). Third, the form of mannosebinding protein found in porcine lymphocytes seems to be related to the liver form (MBP-C) of the rat mannosebinding protein (Childs et al., 1990) as indicated by its native molecular mass and highly specific binding of all oligosaccharides possessing terminal N-acetyl-o-

A

I3

‘ Atdolase \Native lectin l Catalase \ \. Ferritin

log Mr

protein by gel filtration on Sephacryl S-300 (1 x 100 cm) BSA at a flow rate 3Oml/hr.

glycosamine residues. Finally, good correlation was observed for the results obtained by equilibrium methods while the values calculated from the precipitation assay have been lower. This fact is important to consider when evaluating results obtained by modern rapid binding tests relying on fast ~ltration assays or HPLC affinity techniques, which usually work under non-equilibrium conditions. Figure 3 compares the inhibition data from experiments performed with native molecules and the carbohydrate-binding domain. It is considered to be rather typical for C-type lectins that they bind their ligands much more strongly in the highly aggregated state in which several domains may co-operate with their weaker individual binding activities (Piskarev et al., 1990b; Lee et al., 1991b; Taylor et al., 1992). In conclusion, in this paper we have characterized some molecular properties of the mannan-binding peripheral lectin of porcine lymphocytes. It seems that many of its characteristics are very similar to those found for

c

-

94

-

67

-

43

a*,

Fig. 2. Analysis of porcine lymphocyte mannan-binding protein (A) and its collagenase fragment (B) by SDS el~trophoresis. Mofecular weights of marker proteins (C) are given in kDa.

1444

Table 2. Apparent

Oligosaccharide”

association constants (K,) for the interaction between porcine lymphocyte mannan-binding protein and defined oligosaccharides as determined by different methods PEG precipitation assay

Gel filtration

24kZ.I 202 1.3 1.5+ 1.0 13 f 0.84 10 + 0.55 6.4 1: 0.43 0.15 It: 0.01 0.04 If: 0.01 18 + 0.35 I4 F: 0.27 12 f 0.36 9.1 kO.11 29 rt 0.24 15 * 0.38 10 It 0,58 8.3 1: 0.45 4.4 * 0.2 1 2.1 rf: 0.14

72 + 3.5 64 f 3.8 58 rt 4.9 43 * 2.2 30 * 3.1 23 + 1.8 0.62 f 0.03 0.19 + 0.02 55 + 3.4 43 + 4.0 35 f 2.5 20 + 1.3 49 * 2.2 20+ 1.8 37 t 2.2 15k2.3 26 f 2.0 19 + 1.4

M 3.1 BH M4.1 BH M5.1 BH M 6.1 BH M 7.1 BH M 8.1 BH M 3.1 01 M 8.1 01 H 4.3 BH H 5.1 BH H 5.3 BH H 5.4 BH 200.1 BH 201.1 BH 300.1 BH 301.1 BH 400.1 BH 5OO.f BH

“Qligosaccharides (Table 1) were used as Bolton-Hunter hMean + SD of at least three experiments.

tog [cot-s. inhib. (Mf]

X, at 4°C (x 10m6I/rn01)~ Equilibrium Frontal dialysis affinity chromatography 75,4.1 70 + 5.5 55 * 4.3 48 * 4.2 35 _t 4.8 28 + 2.7 0.54 rt 0.04 0.23 + 0.01 48 &-5.0 41 f 5.5 30 2 2.3 30 _t 2.7 53 zt 3.3 15 + 1.8 42 k 3.4 14* 1.1 27 + 2.0 202 1.1

81 * 3.9 69 * 5.4 63 + 6.5 40 zt 4.0 39 r’s4.3 25 _t 3.6 0.58 f 0.02 0.15 rto.02 4Ok4.1 54 24.9 37 &-4.6 27 f 3.5 45 + 5.4 23 rf: 0.7 461 1.6 10 2 0.6 33 zt 2.4 IS & 0.7

reagent derivatives (BH) or reduced substances (01).

Ashwell C. and Harford J. (1982) Carbohydrate-specific receptors of the liver. A. Reo. Biochem. 52, 537-554. Benson B., Hawgood S., &hilling J., elements J., Danim P., Cot-deli B. and White R. T. ($985) Structure of canine pulmonary surfactant a~protein: cDNA and complete amino acid sequence. Proc. nati. Acod. Sci. U.S.A. 82,

6379-6381.

tog [cont.

inhib.

(MI]

Fig. 3. Inhibition of 1251-M3.1-BH binding to native porcine lymphocyte manna-binding protein (A) and its collagenase fragment (B) by high mannose oligosaccharides. m, M3.101; 0, M8.lof; f-J, M3.iBH; 8, MS.IBH. other mammalian mannose lectins, although the fine oligasaccharide specificity of those molecules originating from lymphatic tissues (Kawasaki et al., 1980) has not been reported. The detailed knowledge of the fine oligosaccharide

specificity of this lectin represents one aspect ~portant for better understanding of its multiple biological and immunological functions. Other questions still open for the future research include the lo~ali~tion of the site of biosynthesis of this mannose lectin (hepatocytes, lymphocytes, still other tissues) and the mechanism ofits attachment to the surface of peripheral lymphocytes. Current experiments going on in our laboratories are aimed to address some of these questions.

Bevilacqua M. P., Stengelin S., Gimbrone M. A. and Seed B. (1989) Endothelial leukocyte adhesion molecule 1: an inducible receptor for ~eutrophi~s related to comptement regulatory proteins and lectins. Science 243, If60-I 165. BezouSka K., Crichlow G. V., Rose J. M., Taylor M. E. and Drickamer K. (1991) Evolutionary conservation of intron position in a subfamily of genes encoding care bohydrate-recognition domains. J. biof. Chem. 266, 11604-l 1609. BezouSka K., T&borsk$ O., Kubrycht J., PospiSiI M. and Kocourek J. (1985) Car~hydrate-stricture-de~ndent recognition of desialylated serum glycoproteins in the liver and leucocytes. Two complementary systems. Biochem. J. 227, 345-354. BezouSka K., T&borsk$ O., Kubrycht J., PospiSil M. and Kocourek J. (1990} Identification of porcine lymphocyte membrane lectins as possible NK celi receptors using new derivatives of N-linked oligosaccharides. LPctins &of. Biothem. C&a. Biochem. 7, 207-215. Brownell. M. D., Colley K. J. and Baenziger J. U. (1984) Synthesis, processing and secretion of the core-specific lectin by rat hepatocytes and hepatoma cells. J. bioL Chem. 259, 3925-3932. Ceccarini C., Lorenzoni P. and Atkinson P. H. (1984) Determination of the structure of ovalbumin glycopeptide AC-B by ‘H-nuclear magnetic resonance spectroscopy at 500 MHz. J. mofec. &of, 176, 161-167,

C~amc~rizat~on

of porcine ma~uan-binding

Childs R. A., Fe& T., Yuen C.-T., Driekamer K. and Quesenberry M. (1990) Differential recognition of core and terminal portions of oligosaccharide ligands by carbohydrate-recognition domains of two mannose-binding proteins. J. &al. Chem. 265, 20770-20777. Colley K. J. and Baenziger J. U. (1987) Identification of the post-translational modi~~tions of the core-specific lectin. Hydroxy-proline, hydroxylysine and glucosylgalactosylhydroxylysine residues. J. biol. Chem. 262, 10290-10295. Colley K. J., Beranek M. C. and Baenziger J. U. (1988) Purification and characterization of the core-specific lectin from human serum and liver. Riuchem. J. 256, 61-68. Drickamer K. (1988) Two distinct classes of carbohydraterecognition domains in animal h&ins. J. biool.Chem. 263, 9557-9560. Drickamer K., Dordai M. S. and Reynolds L. (1986) Mannosebinding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. Complete primary structures and homology with pulmonary surfactant apoprotein. J. biol. Chem. 261, 68786887. Drickamer K. and McCreary V. (1987) Exon structure of a ma~nan-birding protein reflects its evolutional reiations~p to the asialoglycoprotein receptor and ~on~brill~ collagen. J. biol. Chem. 262, 2582-2589. Ezekowitz R. A. B. (1991) Anti-antibody immunity. An inlluenza virus mutant has provided new evidence that mannosebinding proteins act as a primitive form of defence against pathogens. Curr. Biol. 1, 60-62. Ezekowitz R. A. B., Day L. E. and Herman G. A. (1988) A h~rna~ ma~nose-binding protein is an acute phase reactant that shares sequence homology with other vertebrate &tins. J. exp. Med. 167, 1034-1046. Ezekowitz R. A. B., Kuhlman M., Groopman J. E. and Byrn R. A. (1989) A human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus. J. exp. Med. 169, 185-196. Ezekowitz R. A. B. and Stahl P. D. (1988) The structure and function of vertebrate mannose lectin-like proteins, J+ Ceil Sci, 9, 121-133. Giorda R., Rudert W. A., Vavassori C., Chambers W. H., Hiserodt J. C. and Trucco M. (1991) NKR-Pl, a signal transduction molecule of NK cells. Science 249, 1298-1300. Grant D. A. W. and Kaderbhai N. (1986) A reassessment of the assay for the asialoglycoprotein receptor and its use in the quantification of receptor distribution in hepatocytes. Biochem. J. 234, 131-137. Huang C.-C., Mayer H. E. and Montgome~ R. (1970) Microheterogeneity and paucid~s~~ity of glycoproteins. Part 1. The carbohydrate of chicken ovalbumins. Carbohydr. Res. 13, 127-137. Hummei J, P. and Dreyer W. J. (1962) Measurement of protein binding phenomena by gel filtration. Biochim. Biophys. Acta 63, 530-532. Ikeda K., Sannoh T., Kawasaki N., Kawasaki T. and Yamashina I. (1987) Serum lectin with known structure activates complement through the classical pathway. J. biol. Chem. 262, 7451-7454. Ikuta K., Takami M., Kim C. W., Monjo T., Myioshi T., Tagaya Y., Kawabe T. and Yodoi J. (1987) Human lymphocyte Fc receptor for IgE. Sequence homotogy of its cloned cDNA with animal lectins, Proc. n&n. Acud. Sci. U.S.A. 84, 819-824. Johnston G. I., Cook R. G. and McEver R. P. (1989) Cloning

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