ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 298, No. 1, October, pp. 49-55, 1992

Ligand Recognition by Purified Human Mannose Receptor Vladimir

K&ry,l,*

JiEi J. F. K?epinsk$,t

Christopher

D. Warren,*

Peter Capek,§

and Philip

D. Stahl*

*Department of Cell Biology and Physiology, Washington University School of Medicine, Box 8228, 660 S. Euclid Avenue, St. Louis, Missouri 63110; TDepartment of Molecular and Medical Genetics, and Carbohydrate Research Centre, University of Toronto, Toronto, Ontario, Canada M5S lA8; SCarbohydrate Unit, Lovett Laboratories, Massachusetts General Hospital, Charlestown, Massachusetts; and $Institute of Chemistry, Slovak Academy of Sciences, Dtibravska 9, 84238 Bra&lava, Czechoslovakia

Received January

27, 1992, and in revised form May 13, 1992

In this work we examine the carbohydrate binding properties of human placental mannose receptor (HMR) using a rapid and sensitive enzyme-linked immunosorbent microplate assay. The assay is based on the inhibition of binding of highly purified receptor to yeast mannan-coated 96-well plates. The specificity of ligand binding was inferred from the potency of different saccharides in blocking HMR binding to the mannan-coated wells. The relative inhibitory potency of monosaccharides was L-Fuc > D-Man > D-Glc > D-GlcNAc > Man-6P + D-Gal % L-Rha + GalNAc. The inhibitory potency of mannose increased by two orders of magnitude when linear oligomers were used. Oligomers containing a-l3- and cr-1-6-linked mannose residues were more inhibitory than those containing (r-1-2and a-1-4-linked mannoses. Linear or branched oligomannosides larger than three units did not have a significant influence on their inhibitory potency; rather, potency was found to decrease in comparison with oligomannosides with three units. Compared to linear oligomers, inhibition of binding was the best using branched mannose oligosaccharides, a-p-Man-bovine serum albumin conjugates, or mannan. A model is discussed in which branched ligand is bound to spatially distinct sites on the HMR. o 1992 Academic Press,

Inc.

Receptor-mediated endocytosis is a major route for protein or glycoconjugate ligand transport into mammalian cells (1). The mannose receptor of macrophages mediates the endocytosis of glycoproteins bearing mannose or fucose oligosaccharide(s) (2,3). The mannose receptor has been isolated from the mouse macrophage cell i To whom correspondence

should be addressed.

0003.9861/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reuroduction in anv form

line 57743 (4), rat liver (11, 12), rabbit alveolar macrophages (5), and human placenta (8). The receptor is a 175-kDa membrane protein requiring Ca2+ for the ligand binding. It efficiently binds mannose-bearing glycoproteins or mannose neoglycoconjugates such as mannosylated bovine serum albumin or lactoperoxidase. Binding occurs with a pH optimum of about 7. Recently, the human macrophage and placental mannose receptors were cloned and from the clones the complete amino acid sequence was deduced (6, 7). The sequence indicates that the receptor is a type I transmembrane protein. The extracellular portion of the receptor contains a 137-amino-acid segment consisting of fibronectin type II repeats followed by eight C-type carbohydrate-recognition domains. These are homologous with the C-type carbohydrate-recognition domains of the asialoglycoprotein receptor, mannose-binding proteins, and other Ca2+ dependent animal lectins (38). Human mannose receptor (HMR)2 from placenta was originally isolated by Lennartz et al (8). The authors showed that it has the same properties as the mannosespecific endocytosis receptor of human monocyte-derived macrophages (9) and rat alveolar macrophages (10-12). Apparent identity of the placental receptor with the macrophage receptor (8) and the ease of isolating large amounts of the former allowed for the present study. The aim of this work was to investigate ligand recognition by purified, homogeneous HMR. An ELISA microplate assay was developed for this purpose enabling us to accurately quantify the inhibitory potency of a large number of sugars, oligosaccharides, and assorted glyco’ Abbreviations used: HMR, human mannose receptor; ELISA, enzyme-linked immunosorbent assay; SDS, sodium dodecyl sulfate; BSA, bovine serum albumin; GlcNAc, N-acetylglucosamine; MBP, mannosebinding protein. 49

Inc. reserved.

KERY

50

ET AL.

conjugates. A comparison of these results with those obtained using a whole-cell assay and the specificity of the receptor, the size of the carbohydrate binding site, and a mechanism accounting for the high-affinity binding of polymeric ligands are discussed. MATERIALS

AND METHODS

HMR was prepared from fresh human placenta obtained from Jewish Hospital, St. Louis, Missouri, as previously described (8). Briefly, placental membranes were washed and extracted with 1% v/v Triton X100. The extract was then subjected to affinity chromatography on mannose-Sepharose. After SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions a single band was observed corresponding to a molecular weight of 175 kDa. Oligosaccharides 19, 20, 24-36 (Table I) were synthesized as reported (26-34). Oligosaccharides 18 and 22 (Table I) were purified from the urine of sheep with swainsonine toxicosis and from pancreatic tissue (23) of the same sheep according to the published procedures (35). Mannose oligosaccharides 16,17, and 2 1 were isolated by acetolysis of the yeast cell wall mannan of Saccharomyces cerevisiae as described (13). This procedure selectively cleaves o-1-6 linkages of the main backbone of mannan and releases manno-di-, tri-, and tetrasaccharides which were subsequently separated by Bio-Gel P2. The structures of all the oligosaccharides used were confirmed by nmr spectroscopy. Yeast mannan was isolated from S. cereuisiae and purified as described (14). This mannan (60 kDa) does not contain any protein impurity. Alkaline phosphatase-conjugated rabbit antigoat IgG, dimannosides, monosaccharides, and ol-D-mannopyranosylphenylisothiocyanate were purchased from Sigma (St. Louis, MO). The concentration of carbohydrates in stock solutions was determined by a resorcinol-sulfuric acid microplate method (16). Bovine serum albumin (BSA), fraction V, which was used for microplate blocking was purchased from Fisher. Contaminating protease activity in the BSA was destroyed by heating at 60°C for 2 h. BSA for glycosylation was purchased from ICN Biomedicals, Irvine, California (purity > 98%). Contaminating carbohydrates in the BSA stock samples were destroyed by periodate oxidation in 0.01 M sodium periodate, pH 5.0, overnight at room temperature following by 24 h dialysis against distilled water at room temperature. a-Man-BSA (65 mol mannose/mol protein) was prepared by the method of Monsigny et al. (15). Mannose and protein content was determined by the resorcinol-sulfuric acid method (16) and Bradford’s protein assay (17), respectively. Costar strip polystyrene mi-

cl0

200

400 600 600 HMR (nglwell)

1000

FIG. 2. HMR binding curve. Yeast mannan (10 pg/well) was coated overnight at room temperature. HMR was bound to the microplate in loading buffer for 1 h at 37’C. The negative control (wells in which HMR binding was inhibited by 1 mg/ml mannan) was sub&acted from all curves presented. The negative control 1 mg/ml mannan + HMR was subtracted from all curves presented.

croplates from Cambridge, Massachusetts, were used in HMR binding, protein, and carbohydrate assays. The goat antiserum against HMR was prepared by standard methods. HMR microplate binding was carried out in the loading buffer (0.01 M Tris-HCl, 0.015 M CaCI,, 1.25 M NaCl, pH 7.4) according to the procedure described in Fig. 1 with subsequent washing (three times) with 0.05% Tween 20 in loading buffer between each step of the procedure. The assay was carried out as follows (all additions were 100 al): 1. Plates were coated with yeast mannan (100 pg/ml) in loading buffer at room temperature overnight. 2. BSA (1%) in loading buffer was added for 1 h at room temperature. 3. The assay was initiated by adding HMR (10 ag/ml) in 50 ~1 of loading buffer to appropriate inhibitors in 50 al of the same buffer followed by incubation for 1 h at 37°C. 4. Goat anti-HMR antiserum, diluted 1:200 in loading buffer, was added for 30 min, at room temperature. 5. Rabbit anti-goat IgG-alkaline phosphatase conjugate, diluted 1: 2000, was incubated for 30 min at room temperature. 6. p-Nitrophenylphosphate, 1 mg/ml in 1 M diethanolamine, 1 mM MgCl, buffer, pH 9.8, was added to the wells and the plate were incubated for 1 h at 37°C. The plates were read at 405 nm on a microplate reader.

RESULTS PNP

t

I

AP-RaG

A I

A I

GaHMR

HMR

Mannan FIG. 1. HMR binding assay in loading buffer. GaHMR, goat antimannose receptor antibodies; AP-RaG, alkaline phosphatase-labeled rabbit anti-goat antibodies; PNP, p-nitrophenylphosphate.

Microplate assay. A solid phase microplate binding assay was developed to examine the affinity of HMR for various ligands (Fig. 1). As shown in Fig. 2, HMR binds to mannan-coated microplates with high affinity. As little as 10 ng of HMR can be detected by an indirect ELISA using a goat anti-HMR serum followed by the conjugate alkaline phosphatase-rabbit anti-goat IgG. A saturating concentration of HMR (0.5 pg/well) employed in the assay corresponded to an absorbancy of 1.5/h at 405 nm. The negative controls were the wells incubated with HMR in the presence of 1 mg/ml mannan that gave the same absorbency as the wells in which the HMR was omitted. The relative slope of the assay, evaluated from 10 identical measurements, varied by less than 15%. Inhibition of HMR binding by monosaccharides. A variety of mono- and oligosaccharides were used to study the carbohydrate binding specificity of the HMR (Table I). The potency of monosaccharides to inhibit HMR

LIGAND

PURIFICATION

BY PURIFIED

binding to mannan-coated microplates decreased in the following order: L-Fuc > D-Man > D-Glc > D-GlcNAc > D-Man-6-P 9 D-Gal B L-Rha 9 D-GalNAc. At least four different sugars (L-Fuc, D-Man, D-Glc, D-GlcNAc) appear to be recognized specifically by HMR. O-cw-Methyl-mannopyranoside 2 increased inhibitory potency five times. HMR shows a slight cY-anomeric preference as indicated by the comparison of the inhibitory potency of (Y- and p-

HUMAN

MANNOSE

RECEPTOR

51

methylmannopyranosides 2 and 3. A hydrophobic 4methylumbelliferyl substituent in position 1 of the mannose ring (derivative 4) also increases the inhibitory potency substantially. Affinity of HMR for oligomannosides. Further mannosylation at position 2,3,4, or 6 decreased the inhibitory potency of the 1-OMe mannopyranoside compared with the unmannosylated mannopyranoside. The inhibitory

TABLE

I

Concentrations of HMR Inhibitors That Caused 50% Inhibition (ID,,) of the HMR (0.5 pug/well) Binding to the Mannan Saccharomyces ceruisiae (10 pg/well)-Coated Microplates Inhibitor 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

D-Man a-Manl-OMe j.SManl-OMe a-Manl-0(4-methylumbelliferyl) D-Man-6-P L-Rha D-Glc (95% of a and 5% of p anomer) D-GlcNAc D-Gal n-GalNAc L-FUC Manal-GManal-OMe Manal4Manal-OMe Manal--3Manal-OMe Manal-aManal-OMe Manal-2Man Manal-aManal-2Man (65%) Manal-3Manal-2Man (35%) 18. Manal-3Manal-6Man/314GlcNAc Manal

\

19. Manal

6 Manal-OMe

ID,, (PM) HMR

150 13,500 35,000 6,000 13,000 19,000 60,000 3,200 2,000 4,000 2,000 3,200 300 2oo 170

Man614GlcNAc

. /3

Manal-2Manal-2Manal Fucal-GGlcNAcal-OMe . Fucal-2Galfil-OMe Fucal \ 6 26. GlcNAc/314GlcNAc/31-OMe ;;.

Fucal

50

20 CI” (5-500)40%

NI < 1000

\

6 27. GlcNAcfll4GlcNAc/31-O(CH,),COOMe

NI < 1000

28 GlcNAcfll-GGalpl-OMe 29: GlcNAcpl-3Gal61-OMe 30. GlcNAcpl-2Gal61-OMe

NI < 1000 NI < 1000 NI < 1000

GlcNAc61

\

6 Galpl-OMe

CI (5,500)30%

i3 GlcNAcP 1 ‘6

Gal614GalNAc Gal614GalNAc61-OMe Galpl3GalNAc 35. Gal6 14GlcNAc6 13Gal6 1-OMe

Manal-OMe

20.

/3

21. Manal-3Manal-2Manal-2Man Manal

‘6

31.

Mancxl

LpXl

23

20

/3

HMR

Manal

5,500 1,000

woo

ID,, (PM)

Inhibitor

280

< < <
Manal-2Mancul-OMe > Manarl-4Mancul-OMe. The Mancul-2Man derivative 16 containing the free reducing end is about 10 times better at inhibiting HMR binding than the same derivative in which the reducing end was methylated (derivative 15). Therefore, free reducing groups or a conformation of the mannose ring that depends on free-reducing groups may play a significant role in HMR recognition of oligomannosides. Further extension of the mannose oligosaccharide chain length produced smaller increases in inhibitory potency. A mannotriose was the optimal length of mannose oligosaccharide that matched the HMR sugar binding site. In this regard, the sequence Mancul-3Manal-6Man 18 is similar to the mixed mannotriose 17 (Mancul-2Manal2Man or Mancul-3Mancul-2Man). The mannotetraose 2 1 had a decreased inhibitory potency compared with the linear mannotrioses. HMR favors binding to branched mannose oligosaccharides. When the a-1-3, l-6 branched trimannoside 19 was used in the HMR binding assay, it was about a lofold more potent inhibitor than the linear trimannosides 17 and 18. A nonspecific substitution such as Lyx in the a-1-3 branch increased the IDS,, 25-fold (derivative 19 versus 20). This indicates that the recognition of the branched mannose oligosaccharide by HMR is highly specific with respect to the mannose residue. However, increasing the number of branches (mannose pentasaccharide 22, Table I) or the length of the branch (mannose pentasaccharide 23, Table I), even with additional mannose units, shows decreased inhibitory potency when compared with the single a-l-3, l-6 branched mannose trisaccharide. Of course, this is assuming that residues on the reducing end of the ligands make little if any contribution to the binding. HMR, like other lectins, should recognize predominantly configurations at the nonreducing end of oligosaccharide. Branched polymeric inhibitors possess the highest binding affinity for HMR. When highly branched, polymeric mannose-containing inhibitors (e.g., yeast mannan from the cell walls of S. cerevisiae or cr-Man-BSA) were used in the HMR binding assay, the IDS0 decreased by three or four orders of magnitude compared with the oligomeric derivatives (Fig. 3). Trypsinization (0.01 mg trypsin/mg a-D-Man-BSA at 37°C for 12 h, followed by inactivation of the trypsin with phenylmethylsulfonyl fluoride) decreased the inhibitory potency of a-D-Man-BSA by two orders of magnitude (data not shown). Thus, both branching and the polymeric nature of a-Man-BSA are necessary for high-affinity binding to the HMR. NonInhibitory effect of nonmannose oligosaccharides. mannose synthetic oligosaccharides containing Fuc/ GlcNAc on the nonreducing end were used in the assay in the concentration range 5-1000 PM (oligosaccharides

ET

AL.

SO

0 .OOl

.Ol

,1

1

(0

Concentration

100

1000

10000100000

fuM]

FIG. 3. Comparison of potency of mannose derivatives to inhibit binding of HMR to yeast mannan (Soccharomyces cereuisiae). The binding assay conditions used were the same as those described in Fig. 1 and Materials and Methods, except different concentrations of inhibitors were added in 50 pl total volume prior to adding HMR (10 pg/ml). Ml, Manal-OMe; M2, 16 (Table I); M3, 17 (Table I); M4, 21 (Table I).

24-31 in Table I). Oligosaccharide 24 (Fuccul-6 GlcNAcOMe) was found to be an effective inhibitor of HMR binding with an IDh0 comparable to that of the branched trimannoside 19. Other oligosaccharides tested (26-30, 32-36) at concentrations up to 1 mM did not inhibit the binding of HMR to the microplate. However, the linear oligosaccharide FucLul-2Galal-OMe 25 and the branched oligosaccharide GlcNAcPl-3(GlcNAcfil6)Gal/31-OMe 31 inhibited binding to a constant of 40 or 30%, respectively, in the range 5-500 PM. Only selected sequences of Fuc and GlcNAc containing oligosaccharides were able to inhibit the HMR binding to the mannancoated micropiate. DISCUSSION The macrophage mannose receptor is a large-molecularweight membrane glycoprotein apparently responsible for both phagocytosis of microorganisms and receptor-mediated endocytosis of mannose glycoconjugates. The recent cloning of the receptor has revealed the unexpected presence of eight putative ligand binding sites, raising the possibility that the broad specificity of the receptor may be accounted for by multiple binding sites (38). As an approach to investigate this question in more detail we developed a rapid, sensitive, and specific HMR-binding assay. Previously, only assays on living or fixed cells have been used to study the receptor-ligand binding and specificity (18). Binding and uptake of radiolabeled ligands such as mannose-BSA by intact macrophages have provided important information regarding HMR specificity. Although more physiological, experiments with intact cells introduce an error due to the influence of the cell surface environment or possible interaction of the ligand with other carbohydrate-specific receptors.

LIGAND

PURIFICATION

BY PURIFIED

The HMR assay presented here has several advantages over studies with intact cells. First, it is possible to carry out many assays simultaneously. Furthermore, with the use of the yeast cell wall mannan, a protein-free natural ligand of the HMR, one can rule out the influences of the protein backbone of glycoproteins or artifical glycoconjugate ligands. Moreover, after subsequent extensive washing only the active receptor is detected on the microplate. Finally, using the microplate assay, it is possible to evaluate the direct binding of the receptor to a variety of other ligands simply by coating them onto the microplate surface. There are no data available in the literature concerning the binding of monosaccharides to the purified mannose receptor. Experiments on the carbohydrate-specific adhesion of rabbit alveolar macrophages to mannose polyacrylamide-derivatized microplates suggest that monosaccharides bind to the mannose receptor (19). The authors found the following order of the inhibitory potency of monosaccharides: D-Man = L-Fuc 2 D-GlcNAc = D-Glc $ D-Gal = L-Rha. Considering the fact that these results were obtained using a whole-cell assay under conditions where other macrophage carbohydrate-specific receptors or other nonspecific interactions might influence the binding, it is striking that our data (i.e., L-Fuc > DMan > D-Glc > D-GlcNAc > D-Man-6-P $ D-Gal $ LRha 9 D-GalNAc) and theirs are quite similar. It has been shown that the closest relative of the mannose receptor, the serum mannose-binding protein, reveals relaxed binding specificity. The mannose-binding protein interacted weakly with most cyclic polyols or sugars but displayed a much higher mannose specificity if glycosides were used in the assay (36). Therefore, the specificity of HMR toward monosaccharides with free reducing ends should also be interpreted with caution. The relative preference of HMR for the a-anomer of Manl-OMe compared with the P-anomer was predictable given the rarity of the occurrence of the ,&Man anomer in natural ligands. The considerable increase in the inhibition of HMR binding by the or-anomer in which the hydrophobic substituent 4-methylumbelliferyl was substituted at position 1 of the carbohydrate ring suggests that high-affinity binding may involve hydrophobic interactions. This argument is supported by the fact that mannobiose (Manal-2Man) is bound to HMR with half the affinity as the 4-methylumbelliferyl derivative. Binding of ““1-a-Man-BSA to whole macrophages was reported to be inhibited by mutant mannans isolated from yeast cells having defects in specific mannosyl transferases (18). The mannose units in these mutant mannans were connected only by a-1-2, a-1-3, a-1-4 or a-l-6 bonds. In whole cells, the order of inhibitory potency of mannans l-6 > l-3 = l-2 > l-4 is similar to the order measured by our assay where l-6 = l-3 > l-2 > l-4. It is interesting to note that natural mannan in yeast cell walls consists predominantly of (u-1-6- a-1-2, and cu-1-3-linked man-

HUMAN

MANNOSE

RECEPTOR

53

noses (13) which are recognized by HMR to a higher degree than a-1-4-linked mannooligosaccharides. This is consistent with the hypothesis that the HMR plays an important role in nonimmunogenic host defense (2, 20, 21). The binding affinity increases considerably in the case of branched mannose-containing ligands with a-1-6, 01l-3 linkages as in structure 19. Increasing the number of mannose units in the oligosaccharides from 3 to 4 or 5 had little influence, and perhaps even decreased their inhibitory potency compared to that of the mannotrioside. However, when an artificially synthesized permannosylated lysyl-serine decapeptide containing five mannose residues was used in the assay (Diaz et al., 1992, in preparation) an approximately one-order-higher IDS0 was measured compared with the pentamannose oligosaccharides 22 and 23. This suggests that highly branched mannose ligands produce a good “fit” for the mannose binding site and that high-affinity binding derives from specific multipoint attachment of the ligand to the receptor molecule at spatially distinguishable binding sites. Further support for the role of multipoint attachment of ligand to the receptor comes from the dramatic increase in inhibitory potency observed for polymeric ligands. While the inhibitory potency of monomers and oligomers is in the millimolar and micromolar ranges, respectively, that of polymeric ligands such as mannans and a-ManBSA is in the nanomolar range. Our observed values of inhibitory constants for the polymers are in agreement with both whole-cell binding experiments (18, 19) as well as receptor immunoprecipitation assays (4). Because increased chain length of both branched and linear mannose oligosaccharides does not have any substantial influence on inhibitory potency, the dramatic increase in inhibitory potency of polymeric and branched ligands is probably due to their simultaneous multipoint attachment to the HMR molecule. The decrease of the inhibitory potency of a-Man-BSA after the trypsin treatment further supports this hypothesis. Further confirmation of this view has appeared recently in studies by Taylor et al. (38). These investigations expressed individual carbohydrate binding domains and compared their affinity for the monomeric and polymeric ligands. The authors also found that domain 4 possesses the highest affinity of all eight domains but alone cannot account for the binding of the receptor to glycoproteins. At least three domains (4, 5, and 7) appear to be required for high-affinity binding and endocytosis of multivalent ligands. Our results show that only particular sequences of nonmannose oligosaccharides are able to bind to the HMR mannose binding site effectively. Oligosaccharides with Fuc/GlcNAc on the nonreducing end except derivative 24 were ineffective as inhibitors, suggesting that Fuc/ GlcNAc ligands can compete with mannose at higher concentrations. Inhibitors 25 and 31 showed some inhibitory effect but only to 30-40% of total receptor bind-

54

KERY

ing. This might be explained by restricted multiple specificity of some of the mannose binding sites. If the simultaneous multiple binding takes place in the attachment of HMR to polymeric mannan, it is possible that not all spatially distinguished carbohydrate binding domains have the same specificity and corresponding affinity for different carbohydrates. High inhibitory potency of linear Fuccul-GGlcNAcLwl-OMe 24, which is comparable with branched trimannoside 19, as well as the inhibitory constants for L-Fuc, GlcNAc, and D-Glc, which are comparable with those for D-Man, suggests that at least some of the receptor carbohydrate binding domains have multiple specificities. This suggestion is supported by the observation that domain 4 recognizes different carbohydrates in a similar order and affinity as the intact receptor (38). Other domains lack the multiple specificity and have lower affinity to the ligands. However, the existence of multiple forms of HMR in the same preparation is also possible. The carbohydrate-recognition domain of HMR is structurally closest to that of hepatic mannose-binding protein C from mammalian liver. The mannose-binding protein has been cloned (6, 7). The sugar binding properties of the carbohydrate-recognition domain of the mannose-binding protein have recently been studied using a fusion protein produced by expression of a cDNA clone in Escherichia coli (23-25). The results indicate that the primary interaction of the binding domain is with the dimannosyl core of complex N-linked oligosaccharides. Additional ligands bound to the domain include terminal fucose of glycoconjugates and peripheral mannose residues of high-mannose-type oligosaccharides. These results are in close agreement with our study of the specificity of the intact HMR. Another mammalian lectin that shares about 60% identity in amino acid composition with the mannose receptor is the serum mannose-binding protein (MBP) (37). The specificity of MBP was recently investigated by use of its carbohydrate-recognition domain expressed from a cloned cDNA (36). The authors used an assay in which 1251-MBP was incubated with yeast cells and the extent of its binding in the presence of different ligands was estimated from the radioactivity associated with the pelleted cells. After comparing HMR with MBP it was striking that HMR showed a much larger difference between the affinity of reducing and nonreducing mannose derivatives than MBP. While IDS0 for HMR varies from 5.5 mM (DMan), to 1 mM (cY-Manl-OMe), to 0.3 mM (Manal2Man), the IDS0 measured for the same ligands for MBP spans a narrow range: 1.3, 1.1, and 1.0 mM, respectively. Thus, while both lectins share specificity to different monosaccharides, the concentration range in the case of HMR varies considerably. This supports the suggestion that serum mannose-binding protein and mannose receptor have potentially sepatate roles (25). However, it is still unknown how much similarity in function there is

ET

AL.

between the mannose receptor and the mannose-binding protein. In conclusion, HMR is able to specifically recognize multiple carbohydrates. For high-affinity binding, the ligands require multiple attachment to spatially distinguishable binding sites. A single mannose binding site may recognize three mannose units in either a linear or branched chain. It remains to be determined whether the multiple specificity of binding is due to the multiple specificity of HMR carbohydrate binding domains or multiple receptor forms with distinguished specificities are present in the same cell. ACKNOWLEDGMENTS The authors thank Dr. James M. Lenhard and Dr. Michael Koval for critical reading of the manuscript. We also thank Elizabeth M. Peters for preparation of goat anti-HMR sera.

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2. Stahl, P. D. (1990) Am. J. Respir. Cell. Mol. Biol. 2, 317-318. 3. Ashwell, G. and Harford, J. (1982) Annu. Reu. Biochem. 51,531554. 4. Blum, J. S., Stahl, P. D., Diaz, R. D., and Fiani, M. L. (1991) Carbohydr. Res. 213,145-153. 5. Wileman, T. E., Lennartz, M. R. and Stahl, P. D. (1986) Proc. N&l. Acad. Sci. USA 83,2501-2505. M. E., Conary, J. T., Lennartz, M. R., Stahl, P. D., and Drickamer, K. (1990) J. Biol. Chem. 265, 12156-12162.

6. Taylor,

I. Ezekowitz,

A. B., Sastry, K., Baily, Exp. Med. 172,1785-1794.

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M. R., Cole, F. S., Shepherd, V. L., Wileman, Stahl, P. D. (1987) J. Biol. Chem. 262, 9942-9944.

A. (1990) J. T. E., and

9. Shepherd, V. L., Campbell, T. J., Senior, R. M., and Stahl, P. D. (1982) J. Reticuloendothel. Sot. 32, 423-431. 10. Wileman, T. E., Lennartz, M. R., and Stahl, P. D. (1986) Proc. Natl. Acad. Sci. USA 83,2501-2505. 11. Haltiwanger,

R. S., and Hill, R. (1986) J. Biol. Chem. 261, 7440-

7444. R. S., and Hill, R. (1986) J. Biol. Chem. 261, 1569615702. 13. Kocourek, J., and Ballou, C. E. (1969) J. Bacterial. 100,1175-1181. 14. Kogan, G., Pavliak, V., and Masler, L. (1988) Carbohydr. Res. 172,

12. Haltiwanger,

243-253. 15. Monsigny,

M., Roche, A.-C., and Midoux,

P. (1984) Biol. Cell. 51,

187-196.

16. Monsigny, M., Petit, C., and Roche, A.-C. (1988) Anal. Biochem. 175,525-530. 17. Bradford, M. (1976) Anal. Biochem. 220, 665-675. 18. Stahl, P. D., Rodman, J. S., Miller, M. J., and Schlesinger, P. H. (1978) Proc. Natl. Acad. Sci. USA 75, 1399-1403. 19. Largent, B. L., Walton, K. M., Hoppe, C. A., Lee, Y. C., Schnaar, R. L. (1984) J. Biol. Chem. 259, 1764-1769. 20. Ezekowitz, R. A. B., Kuhlman, M., Groopman, J., and Byrn, R. A. (1989) J. Exp. Med. 169, 185-196. 21. Ezekowitz, R. A. B., and Stahl, P. D. (1988) J. Ceil Sci. S(Suppl.), 121-133.

LIGAND

PURIFICATION

BY PURIFIED

22. Kogan, G., Pavliak, V., Sandula, J., and Masler, L. (1991) Carbohydr. Polym. 14,65-76. 23. Childs, R. A., Drickamer,

K., Kawasaki, T., Thiel, S., Mizouchi, and Feizi, T. (1989) Biochem. J. 262,9557-9560.

T.,

24. Drickamer, K. (1989) Biochem. Sot. Trans. 17, 13-15. 25. Childs, R. A., Feizi, T., Yuen, C.-T., Drickamer, K., and Quesenberry, M. S. (1990) J. Biol. Chem. 265,20770-20777.

26. Winnik,

J. J. F.

27. Winnik,

J. J. F., (1982) J. Org.

F. M., Brisson, J. R., Carver, J. P., and Kfepinskjr, (1982) Carbohydr. Res. 103, 15-28.

F. M., Carver, J. P., and Krepinskjr, Chem. 47, 2701-2707.

28. Schwartz, D. A., Lee, H. H., Carver, J. P., and Kiepinsky,

35.

J. J. F. (1985) J.

37.

pinsky, J. J. F. (1986) Can. J. Chem. 64, 1912-1918.

RECEPTOR

68,953-957. 34. Lee, H. H., Congson, L. N., Whitfield,

36.

30. Lee, H. H., Schwartz, D. A., Harris, J. F., Carver, J. P., and Kie-

MANNOSE

55

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