The C N-linked glycan is a critical component of the ligand for the erythrocyte BAEBL

D. C. Ghislaine Mayer*†, Lubin Jiang*, Rajeshwara N. Achur‡, Ikuko Kakizaki‡, D. Channe Gowda‡§, and Louis H. Miller*§

*Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852; and ‡Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA 17033

Contributed by Louis H. Miller, December 22, 2005 Plasmodium vivax uses a single member of the Duffy binding-like more likely to recognize oligosaccharides than on (DBL) receptor family to invade erythrocytes and is not found in glycophorin C or on other . Indeed, the extracellular West Africa where its erythrocyte ligand, the Duffy group portion of glycophorin C has no homology with any other , is missing. In contrast, Plasmodium falciparum expresses in the human database. Furthermore, treatment of erythrocytes four members of the DBL family, and remarkably, single-point with , proteinase K, or could not abolish the mutations of two of these receptors (BAEBL and JESEBL) bind to binding of BAEBL (INKK) to these enzyme-treated erythro- entirely different erythrocyte ligands, greatly expanding the range cytes (8), suggesting that the ligand is either a glycolipid or a of erythrocytes that P. falciparum can invade. In this article, we highly protease resistant . Thus, a more viable describe the molecular basis of the binding specificity for one alternative to a ligand is the possibility that the N-linked BAEBL variant (VSTK) that binds to glycophorin C. We demonstrate oligosaccharide either alone or in combination with O-linked that soluble glycophorin C completely blocks the binding of BAEBL oligosaccharides provides the basis for the diversity of erythro- ␮ (VSTK) to human erythrocytes, requiring 0.7 M for 50% inhibition, cyte ligands recognized by the BAEBL variants. a concentration similar to that required by to block We studied the inhibition of binding of the BAEBL variant the binding of erythrocyte-binding antigen 175 to erythrocytes. that fails to bind Gerbich-negative erythrocytes and compared it BAEBL (VSTK) does not bind to Gerbich-negative erythrocytes that with those that bind Gerbich-negative erythrocytes using puri- express a truncated form of glycophorin C because it lacks 3. fied intact glycophorin C. We found that the only BAEBL variant The N-linked oligosaccharide of Gerbich-negative glycophorin C has a markedly different composition than the wild-type glycoph- that could not bind Gerbich-negative erythrocytes was inhibited orin C. Moreover, removal of the N-linked oligosaccharide from the by intact glycophorin C. Removal of the N-linked oligosaccha- wild-type glycophorin C eliminates its ability to inhibit binding of ride from glycophorin C abolished the inhibition. Furthermore, BAEBL (VSTK) to erythrocytes. These findings are consistent with we demonstrate that the N-linked oligosaccharide on glycoph- the ligand for BAEBL (VSTK) being, in part, the N-linked oligosac- orin C from Gerbich-negative erythrocytes is different in com- charide and suggest that single-point mutations in BAEBL allow P. position compared with the N-linked oligosaccharide on wild- falciparum to recognize oligosaccharides on different erythrocyte type glycophorin C. These findings provide evidence that the surface or glycolipids, greatly increasing its invasion ligand for BAEBL is an N-linked oligosaccharide or a cluster of range. N- and O-linked oligosaccharides and have important implica- tions for the mechanism by which BAEBL mutations are selected mutations ͉ oligosaccharides ͉ DBL family and for the survival of P. falciparum.

Results and Discussion edundancy in erythrocyte invasion pathways is a critical Rfactor in the survival of Plasmodium falciparum. Unlike We previously demonstrated that four of the variants of BAEBL, Plasmodium vivax, which was eliminated from West Africa each differing in a single in region II, displayed a because of the absence of its erythrocyte ligand, the Duffy blood different specificity for erythrocytes as determined by their group antigen (1), there are no known mutations in erythrocyte binding patterns to trypsin- and -treated eryth- surface proteins that lead to refractoriness to erythrocyte inva- rocytes and to Gerbich-negative erythrocytes (4, 6). The region sion by P. falciparum. The protein on the surface of P. vivax that II domain of BAEBL that binds erythrocytes has mutations in binds the Duffy blood group antigen belongs to a family of only four amino acids in 20 clones of P. falciparum sequenced in Plasmodium called the Duffy binding-like (DBL) family (2). from around the world (4). The four amino acids at these P. falciparum expresses four DBL genes compared with a single positions define five BAEBL variants (VSTK, VSKK, ISKK, in P. vivax, greatly expanding the potential receptor-ligand INKK, and INRE). All BAEBL variants with the exception of interactions for P. falciparum (2, 3). In addition, P. falciparum is INKK were studied (4), and only the VSTK variant failed to bind able to recognize different erythrocyte ligands through single- to Gerbich-negative erythrocytes, which expresses a truncated point mutations in the receptor domain (region II) in two of its form of glycophorin C, indicating that glycophorin C is the four DBL genes (BAEBL and JESEBL) (4, 5), further ensuring erythrocyte ligand for the VSTK variant (4). survival. Each clone of BAEBL is identified by four polymorphic amino acids in region II (4). One of the variants, BAEBL (VSTK), binds glycophorin C based on its inability to bind Conflict of interest statement: No conflicts declared. Gerbich-negative erythrocytes that lack exon 3 in glycophorin C Freely available online through the PNAS open access option. (4, 6, 7). Because point mutations in BAEBL region II affect the Abbreviations: DBL, Duffy-binding ligand; Man, mannose. receptor specificity for its ligand on erythrocytes, it is critical to †Present address: Virginia Commonwealth University, 1000 W. Cary Street, Room 126, determine the ligand recognized by BAEBL. Richmond, VA 23284-2012. Is it likely that the BAEBL variants that are so similar in §To whom correspondence should be addressed. E-mail: [email protected] or lmiller@ sequence recognize different peptide backbones as their eryth- niaid.nih.gov. rocyte ligands? It is our view that point mutations in BAEBL are © 2006 by The National Academy of Sciences of the USA

2358–2362 ͉ PNAS ͉ February 14, 2006 ͉ vol. 103 ͉ no. 7 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510648103 Downloaded by guest on September 25, 2021 Fig. 1. Glycophorin C completely inhibits the binding of BAEBL (VSTK) but not other BAEBL variants to human erythrocytes. (A) Immunoprecipitation of BAEBL variants VSTK (Dd2Nm), ISKK (E12), INKK (3D7), and INRE (MCamp) eluted from human erythrocytes following incubation with various concentrations of glycophorin C. (B) The percent inhibition of binding of VSTK by different concentrations of glycophorin C BAEBL to human erythrocytes was obtained separately three times; the inhibition of binding of the other variants by glycophorin C was performed twice. Data are shown as the mean of these experiments, and the error bars are the standard deviation. The intensity of the BAEBL band was measured by using IMAGEJ (http:͞͞rsb.info.nih.gov͞ij͞), and the data are expressed as the percentage of binding of BAEBL to erythrocytes that were not preincubated with glycophorin C. The inhibition of binding of VSTK BAEBL reached 50% at 0.7 ␮M. MICROBIOLOGY

Binding of BAEBL (VSTK) to Erythrocytes Is Inhibited by Glycophorin C. previous study that antibodies to glycophorin C partially inhib- To further analyze the specificity of the BAEBL variants for ited the binding of INKK to erythrocytes (10). However, these glycophorin C, we determined the ability of different concen- results are not entirely conclusive because binding to Leach and trations of soluble glycophorin C to inhibit binding of four of the Yus erythrocytes was performed with erythrocytes frozen as five BAEBL variants to human erythrocytes. To study the effect pellets in liquid nitrogen that may have been unsuitable for of soluble glycophorin C on the binding of BAEBL (VSTK) to the binding assay. In addition, an alternative explanation for the human erythrocytes, the supernatant from P. falciparum partial inhibition by antibody to glycophorin C is that the Dd2(NM)-infected erythrocytes expressing the BAEBL (VSTK) antibodies affected the function of another protein, perhaps was first incubated with purified glycophorin C at a concentra- through band 4.1, which connects glycophorin C to the cytoskel- tion range from 0.3 to 100 ␮M before adding the mixture to eton (11). For example, band 4.1 also interacts with band 3 and erythrocytes. The inhibition, or lack thereof, was determined by CD44 (12). Furthermore, there is evidence that band 3 is one of the quantity of parasite protein bound to erythrocytes. We found the ligands for parasite invasion (13). Our observation that the purified glycophorin C completely blocked the binding of the binding of BAEBL (INKK) to erythrocytes could not be inhib- ited by purified, soluble glycophorin C at a concentration BAEBL (VSTK) to erythrocytes at concentrations of 20–30 ␮M 100-fold greater than that required for blocking of binding of and that 50% inhibition was achieved at a concentration of 0.7 VSTK to erythrocytes strongly suggests that glycophorin C is not ␮M (Fig. 1). Notably, another member of the DBL family, the ligand for BAEBL (INKK). erythrocyte-binding antigen 175, fails to bind to human eryth- rocytes in the presence of soluble glycophorin A in the same Glycophorin A Fails to Inhibit Binding of BAEBL to Erythrocytes. It has concentration range (9). been reported that glycophorin A, in addition to glycophorin C, Three of the BAEBL variants (INRE, ISKK, and INKK) could act as the ligand for BAEBL (VSTK) and BAEBL (INKK) lacked specificity for glycophorin C, showing no inhibition of (7). This suggestion was based on the binding of BAEBLs (VSTK ␮ binding by glycophorin C even at a concentration of 100 M (Fig. and INKK) in a Western overlay assay to the region of the gel 1). These data demonstrate that glycophorin C is the erythrocyte where the dimer of glycophorin A migrates. In contrast, we ligand for only the VSTK variant of BAEBL and is not the ligand found that 100 ␮M of glycophorin A had no effect on the binding for the other three highly related variants. of VSTK, VSKK, ISKK, or INRE to erythrocytes (data not Contrary to our results, Lobo et al. (10) concluded that shown). glycophorin C was the ligand for BAEBL variant (INKK) based on the observation that INKK did not bind to Leach erythrocytes Cleavage of the N-Glycan from Glycophorin C Destroys Its Ability to that lack glycophorin C or to Yus erythrocytes that have a Inhibit Binding of BAEBL (VSTK) to Erythrocytes. What is the bio- deletion of exon 2 of glycophorin C. It was also observed in the chemical nature of the ligand on glycophorin C that BAEBL

Mayer et al. PNAS ͉ February 14, 2006 ͉ vol. 103 ͉ no. 7 ͉ 2359 Downloaded by guest on September 25, 2021 protease contaminant in the N-glycanase. In the case of Gerbich- negative glycophorin C, the mobility of the untreated and N-glycanase-treated glycoprotein corresponded to 31.9 and 29.4 kDa, respectively. The effect of N-deglycosylated glycophorin C on the binding of BAEBL (VSTK) to erythrocytes was determined. The N- deglycosylated glycophorin C was unable to inhibit the binding of VSTK BAEBL to erythrocytes at a concentration Յ25 ␮M, whereas intact glycophorin C tested in parallel completely inhibited the binding in a dose-dependent manner, as described above (see Fig. 3 B and C). Nevertheless, the N-glycans released from glycophorin C were unable to inhibit the binding of glycophorin C to a concentration Յ100 ␮M (data not shown). Taken together, these data demonstrate that the N-linked oli- gosaccharide on glycophorin C was necessary for the binding of BAEBL (VSTK) to glycophorin C, but the isolated N-glycans themselves were not sufficient to inhibit the binding. Fig. 2. Preparation of N-deglycosylated glycophorin C of N-linked glycans for carbohydrate analysis (Table 1). Normal (Upper) and Gerbich-negative The Biochemical Nature of the N-Glycan on Gerbich-Negative Glyco- glycophorin C (Lower) were N-deglycosylated with N-glycanase and chro- phorin C. The above observations raise the question of whether the matographed on Sephadex G-50 columns. Fractions having the released N- N-linked oligosaccharide on Gerbich-negative glycophorin C dif- glycans (fraction numbers 32–41) were combined and dialyzed by using fers from the wild-type glycophorin C. Indeed, we found that the membranes with a 500-Da molecular mass cut-off and lyophilized. The void N-glycan on Gerbich-negative and normal glycophorin C differed volume determined by chromatographing blue dextran (BD) and the elution in their composition (Table 1). The N-linked oligosaccharide from volume of glucose (Glc) are indicated by arrows. erythrocytes with normal glycophorin C consists of NeuNAc͞Gal͞ GlcNAc͞Man in the molar ratios of 1.8:1.9:4.8:3.0 (Table 1). In (VSTK) recognizes? As discussed above, we hypothesized that contrast, the N-linked oligosaccharide of Gerbich-negative eryth- the ligand may be the N-linked oligosaccharide alone or in rocytes contained a markedly elevated proportion of Man (Table combination with O-linked oligosaccharides. To determine 1), suggesting the presence of a considerable amount of high whether the N-linked oligosaccharide on glycophorin C was Man-type structures in addition to sialylated complex-type oligo- critical for binding to BAEBL (VSTK), glycophorin C was saccharides. Thus, glycophorin C of Gerbich-negative erythrocytes treated with protease-free N-glycanase under nondenaturing differed not only by a deletion in exon 3, leading to a truncated conditions, and the products were recovered by size-exclusion protein, but also in the carbohydrate composition of its N-linked chromatography on Sephadex G-50 columns (Fig. 2). Carbohy- oligosaccharide. This difference in the N-linked oligosaccharide drate compositional analysis showed that although untreated and the failure to inhibit erythrocyte binding with N-linked glycophorin C had relatively high levels of mannose (Man) and deglycosylated glycophorin C is consistent with the N-linked oli- GlcNAc, the N-glycanase-treated glycophorin C completely gosaccharide being the ligand for the BAEBL (VSTK) variant. lacked Man and had only a low level of GlcNAc (Table 1), N-glycans encompass a diverse repertoire of polymeric branched indicating complete removal of the N-linked oligosaccharide. In structures initially added to proteins as high Man structures in the contrast, GalNAc quantitatively remained associated with the endoplasmic reticulum and subsequently processed to a variety of N-glycanase-treated glycophorin C, demonstrating that the en- complex structures in the Golgi apparatus (14). Evidence has been zyme did not remove the O-linked glycans. presented from the Robbins Laboratory (Massachusetts Institute of Furthermore, immunoblotting indicated that although un- Technology, Cambridge, MA) that physical accessibility of the treated glycophorin C electrophoresed as a diffuse band with an oligosaccharide affects its processing in the Golgi (15). average molecular mass of 36.5 kDa, the N-glycanase-treated glycophorin C migrated as a somewhat less diffuse band with a The O-Linked Oligosaccharides on Gerbich-Negative Glycophorin C. It molecular mass of 33.7 kDa on SDS͞PAGE (Fig. 3A). This result was found that Gerbich-negative glycophorin C had reduced shows that there was no cleavage of glycophorin C due to a O-linked oligosaccharides compared with wild-type glycophorin

Table 1. Carbohydrate composition of normal and Gerbich-negative glycophorin C before and after N-deglycosylation Sugar composition (relative molar ratios)*

Sample GalNAc GlcNAc Gal Man NeuNAc

Normal glycophorin C 11.4 6.0 13.1 3.0 20.5 N-deglycosylated normal glycophorin C 11.8 1.7 12.1 ND 18.2 N-linked glycans of normal glycophorin C ND 4.8 1.9 3.0 1.8 Gerbich-negative glycophorin C 4.8 2.9 5.8 8.0 6.7 N-deglycosylated Gerbich-negative glycophorin C 5.0 0.2 4.9 ND 5.6 N-linked glycans of Gerbich negative glycophorin C ND 2.5 1.1 7.8 0.9

Untreated glycophorin C, N-deglycosylated glycophorin C, and N-linked glycans released by treatment of glycophorin C with N-glycanase were hydrolyzed with either 2.5 M trifluoroacetic acid (to release neutral sugars and hexosamines) or 2 M acetic acid (to release sialic acid). The hydrolysates were dried, and the sugars were analyzed by high-pH anion-exchange HPLC. ND, not detected. *The sugars in the hydrolysates were quantitated from the chromagraphic peak areas and by using standard sugar solutions.

2360 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510648103 Mayer et al. Downloaded by guest on September 25, 2021 Fig. 3. N-deglycosylated glycophorin C did not inhibit binding of BAEBL (VSTK) to human erythrocytes. (A) Untreated (GPC) and N-glycanase-treated (N-D GPC) normal glycophorin C and Gerbich-negative glycophorin C were analyzed by Western blotting by using anti-glycophorin C antibody. The positions of molecular mass (kDa) marker proteins are indicated. (B) Immunoprecipitation of BAEBL (VSTK) eluted from human erythrocytes preincubated with either glycophorin C (GPC) or N-deglycosylated glycophorin C (N-Deglycos. GPC). The molecular weight standards are shown on the left. (C) The percent inhibition of binding of VSTK BAEBL to erythrocytes by glycophorin C and N-deglycosylated glycophorin C. Data are shown as the mean of two independent experiments, and the error bar is the standard deviation.

C (Table 1). The exon-3 deletion of Gerbich-negative glycoph- represent a mechanism of pathogen evasion by long-lived verte- MICROBIOLOGY orin C does not eliminate any or threonines on the brates such as humans (20). Therefore, microorganisms like the protein to which O-linked oligosaccharides are linked. The influenza virus and P. falciparum may have evolved polymorphisms reduced mol of O-linked oligosaccharides may result from the generated by single-point mutations in their receptors to use the diminished access of the shortened protein to the glycosyltrans- diversity in carbohydrate ligands. ferases in the Golgi, similar to the explanation for the high Man In contrast to BAEBL, point mutations in the P. vivax structure of the N-linked oligosaccharide. Duffy-binding receptor does not lead to invasion by a Duffy blood group independent pathway (21). Because of this require- Concluding Remarks. A question raised by our findings is why the ment, P. vivax is not found in West Africa where most people are purified N-linked oligosaccharide itself could not inhibit binding. Duffy blood group-negative (1). The Duffy-binding receptor of This result is consistent with the finding that glycophorin A, P. vivax binds a peptide on the Duffy blood group proteins, which has a similar N-linked oligosaccharide content (16) to although it also binds to a sulfate group on the peptide with glycophorin C, did not inhibit binding of BAEBL (VSTK) to higher affinity (22, 23). Thus, it is more likely that single-point erythrocytes. One possibility is that the sialic acid on the mutations in the erythrocyte-binding domain of BAEBL that oligosaccharide interacts with positively charged amino acid on bind different glycoproteins recognize as their ligand the vari- the peptide backbone in a way that exposes the ligand. It is also ation in the distribution and carbohydrate composition of the N- possible that clusters of sialic acid residues of N-linked oligo- and O-linked oligosaccharides. saccharides and those of several closely spaced O-linked oligo- saccharide in glycophorin C or a conformational structure Materials and Methods involving residues of both N- and O-linked oligosaccharides P. falciparum Clones Used. The parasite clones used in this study were provide multivalency for efficient binding of BAEBL (VSTK). E12 (ISSK), HB3 (VSTK), Malayan Camp (INRE), 3D7 (INKK), Is there precedent for a single-point mutation in a receptor and Dd2͞Nm (VSTK). of the parasite clones were altering the receptor for oligosaccharide ligands? A single mutation confirmed by microsatellite analysis (24). Parasite culture and DNA in the receptor pocket of the hemagglutinin of the influenza virus isolation were performed as described in ref. 6. changes the receptor to allow recognition of ␣-2,6-sialic acid instead of ␣-2,3-sialic acid (17). Carbohydrates are extremely versatile in Erythrocyte-Binding Assay. Soluble, 35S-labeled parasite proteins structure and composition (18). They are the ligands of choice for were obtained from the culture supernatant of each clone as both prokaryotic microorganisms and P. falciparum (19). Varia- described in ref. 6. 35S-labeled supernatant (100 ␮l) was used in tions in the terminal sequences of oligosaccharides, the linkages of absorption to erythrocytes and their elution, as described in ref. 6 these sequences, as well as unusual modifications found in carbo- with the following modifications. The supernatant was incubated hydrates play important biological roles. Such modifications can with purified glycophorin C in concentrations ranging from 0.3 to mask the recognition of many microorganism receptors (20). It has 100 ␮Mfor1hatroom temperature with rotation before adding been suggested that the diversity of surface glycan molecules an equal volume of erythrocytes. After a 30-min incubation at room

Mayer et al. PNAS ͉ February 14, 2006 ͉ vol. 103 ͉ no. 7 ͉ 2361 Downloaded by guest on September 25, 2021 temperature, the mixture was centrifuged at 14,000 ϫ g, and the at 37 °C for 18 h and chromatographed on a Sephadex G-50 supernatant was discarded. Erythrocytes were resuspended in 500 column (1 ϫ 50 cm) by using PBS, pH 7.2. Fractions (0.66 ml) ␮l of RPMI medium 1640 and rapidly layered onto dibutyl phtha- were collected, and absorption at 280 nm was measured. Ali- late (Sigma). After the mixture was centrifuged at 14,000 ϫ g for 1 quots of fractions were analyzed for sialic acid by the micrope- min in a microfuge, supernatant and oil were removed by aspiration. riodate-resorcinol method (28). Fractions containing glycoph- The Eppendorf tube was turned upside down to remove the orin C and the released N-linked oligosaccharides were pooled residual oil and any remaining supernatant. The erythrocyte pellet separately, dialyzed (the latter by using a 500-Da cut-off dialysis was washed twice with 500 ␮l of RPMI medium 1640, and bound membrane), and lyophilized. parasite proteins were eluted from the erythrocytes with 1.5 M NaCl. The eluate was handled as described in ref. 6, including the Carbohydrate Composition Analysis. Untreated glycophorin C, N- immunoprecipitation of BAEBL. deglycosylated glycophorin C, and N-linked oligosaccharides (10 ␮g of protein or oligosaccharides corresponding to 10 ␮gof Isolation of Total Glycophorin from Erythrocyte Membranes. The protein) were dissolved in 400 ␮l of 2.5 M trifluoroacetic acid preparation of erythrocyte membranes and isolation of total gly- and hydrolyzed at 100°C for 5 h. The hydrolysates were dried in cophorin from the membranes were done as reported in ref. 25. a Speed-Vac (Savant Instruments, Inc., Farmingdale, NY) and Briefly, Ϸ400 ml of human blood obtained from a was analyzed for neutral sugar and hexosamines on a CarboPac PA10 washed with cold isotonic buffer and lysed in 4 liters of hypotonic high-pH anion-exchange column (2 ϫ 250 mm) by using a buffer. The membranes were recovered by centrifugation and then Dionex BioLC HPLC system coupled to pulsed amperometric extracted with 10 mM Tris⅐HCl, pH 7.4, containing 85 mM deoxy- detector. Elution was with 20 mM sodium hydroxide. Response cholate as described in ref. 26. To the extract, an equal volume of factors for the monosaccharides were determined by using ϫ 50% phenol was added, stirred, and centrifuged at 5,200 g. The standard sugar solutions (30). For sialic acid analysis, samples upper aqueous layer containing total glycophorin was collected, were dissolved in 2 M acetic acid (300 ␮l) and hydrolyzed at 80°C dialyzed against water, and lyophilized. The solid mass was sus- for 1 h. The hydrolysates were dried in a Speed-Vac, and the pended in cold 90% ethanol and stirred to remove the residual residue was dissolved in water and analyzed as above by using 100 phenol and recovered by centrifugation. The pellet of pure glyco- mM NaOH͞150 NaOAc as the eluent. phorin C was dissolved in water, dialyzed, and lyophilized. Immunoblot Analysis of Untreated and N-Deglycosylated Glyco- Purification of Glycophorin C. Glycophorin C was purified from phorin C. Untreated or N-deglycosylated glycophorin C (2–3 ␮g total glycophorin as described in ref. 27. The total glycophorin, each) was electrophoresed on 4–15% SDS͞PAGE in the pres- obtained from the procedure above, was dissolved in 25 mM ͞ ence of 2-mercaptoethanol. The protein bands in the gels were phosphate buffer, pH 7.8, containing 25 mM NaCl 0.1% deoxy electrotransferred onto poly(vinylidene difluoride) membranes, cholate. The solution was chromatographed on Bio-Gel A 1.5 ϫ and the membranes were blocked with 5% fat-free dry milk in (2.5 90 cm) in the above buffer. Fractions (4.5 ml) were TBST buffer (50 mM Tris⅐HCl, pH 7.5, containing 150 mM collected, absorption at 260 and 280 mM was measured, and NaCl͞0.05% Tween 20). The membranes were incubated with aliquots were analyzed for sialic acid by the periodate–resorcinol 1:200-diluted mouse anti-glycophorin monoclonal antibody. The method (28). Fractions containing glycophorin C were sepa- bound antibody was detected with 1:2,000-diluted horseradish rately pooled, dialyzed, and lyophilized. peroxidase-conjugated goat anti-mouse IgG and chemilumines- cence substrate (Kirkegaard & Perry Laboratories). Deglycosylation of Glycophorin C by N-Glycanase. N-deglycosylation of glycophorin C was performed by using protease-free PNGase ͞ We thank Dr. Veer P. Bhavanandan for his advice during the preparation F(N-glycosidase F; specific activity of 1,800,000 units mg; New of glycophorin C and Dr. Susan Pierce for carefully reviewing the England Biolabs) purified from Flavobacterium meningosepti- manuscript. Work done in the laboratory of D.C.G. was supported by cum as described in ref. 29. To a solution of glycophorin C (Ϸ2 National Institute of Allergy and Infectious Diseases (NIAID), National mg) in 50 mM sodium phosphate, pH 7.5, was added 100 ␮lof Institutes of Health (NIH) Grant AI45086. This work was also sup- N-glycanase (50,000 units). The reaction mixture was incubated ported, in part, by the intramural research program of NIAID, NIH.

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