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PubMed1985

Resistance to natural killer cytolysis and neutral glycolipid composition of cultured human glioma and fetal brain cells.

Natural killer cells (NK) demonstrate cytotoxicity against a wide range of cultured cells without prior sensitization, and the sensitivity of many target cells to NK attack is altered by exposure to interferon. The structures on the target cells conferring sensitivity or resistance to NK are not known, but glycolipids are suggested to be related to NK susceptibility. To determine possible relationships between target cell glycolipids and NK cytolysis, the effects of human beta-interferon (IFN) on the neutral glycolipid composition and sensitivity to NK cytolysis of cultured cells from four human gliomas and two fetal brains were analyzed. Compared to MOLT-4 and Raji cells all six neural cell lines were quite resistant to NK, and IFN slightly increased this resistance. IFN also caused increases in the amounts of non-hydroxy fatty acid cerebroside, ceramide trihexoside, asialo-GM1, asialo-GM2 and globoside. Increased molar proportions of ceramide tri- and tetra-saccharides occurred in the two glioma lines which had the greatest increases in NK resistance following IFN exposure. It is concluded that neutral glycolipids may play a role in the mechanisms responsible for resistance of some glioma cells to NK cytolysis.

Astrocytoma↗
PubMed1999

Glycolipid markers of astrocytomas and oligodendrogliomas.

Neutral glycolipids and gangliosides were analyzed in 149 astrocytomas (A), 46 oligodendrogliomas (O), and 21 oligoastrocytomas (OA) to determine if specific glycolipids correlate with histologic diagnosis and grade. Positivity for asialoGM1 (GA1) and negativity for paragloboside by immuno-TLC correlated with histological diagnosis of O and OA, whereas the reverse pattern correlated with A. High levels (over 5 microg hexose per mg dry weight) of CMH generally correlated with an O component, but the association was not as strong as for either GA1 presence or paragloboside absence. Pilocytic astrocytomas and pleomorphic xanthoastrocytomas had high proportions (> 15%) of globoside, low ratios (< 0.5) of GD1a: GD1b, and identifiable ceramide trihexoside (CTH). Three gangliosides of the 1b pathway were progressively lost with increasing grade of A, but a similar correlation with grade was not seen in O or OA. A high proportion of cases expressing sialosylparagloboside (3'LM1; 6'LM1) were grade 4 A. Glycolipids are synthesized by glycosyltransferases that add specific sugars to the nascent oligosaccharide. Correlation of specific glycolipids with histological diagnoses and grades indicate that these tumor types express specific patterns of glycosyltransferases, several of which have been cloned. It is possible that critical genes coding for these enzymes are deleted, overexpressed, or mutated in certain tumor types and grades, thus leading to the patterns of glycolipids that we found to be associated with these tumors.

Adolescent↗
PubMed1985

A monoclonal antibody against a Burkitt lymphoma associated antigen has an anti-Pk red blood cell specificity.

The blood group specificity of a rat IgM monoclonal antibody (38-13) directed against most EBV-positive or -negative Burkitt's lymphoma was investigated. The target antigen was previously identified as the neutral glycolipid ceramide trihexoside (CTH), a substance which accumulates in red cells from very rare individuals of the Pk phenotype and which appears as a normal intermediate in the biosynthetic pathway of the P blood group antigen (globoside). The 38-13 antibody agglutinated Pk1 and Pk2 red cells at 4 degrees C with a very high titre but was inactive against native P1, P2 and p erythrocytes, although a very weak activity was noticed towards papain-treated P1 and P2 erythrocytes. These results were confirmed by an indirect radio-binding assay which also demonstrated that the 38-13 antibody reacted with lymphocytes, fibroblasts and EBV-positive lymphoblastoid cell lines derived from Pk1 and Pk2 individuals but not from other donors. These findings demonstrate that the 38-13 monoclonal antibody previously considered as specific of Burkitt cells could be routinely used as an anti-Pk blood group typing reagent. The mechanism of CTH accumulation in Burkitt cells and Pk red cells is probably different and might be associated respectively with the activation of an alpha-4-galactosyltransferase or the genetic blockage of a beta-3-N-acetylgalactosaminyltransferase.

Animals↗
PubMed1978

Abnormal glycolipid composition of erythrocytes with a weak P antigen.

The erythrocytes of a normal man were agglutinated more weakly than normal cells by several anti-P1PPK sera, and exhibited a decreased capacity to absorb these antibodies. Analysis of his erythrocyte glycosphingolipids revealed that the globoside (P antigen) content was less than 25% of normal, and trihexosyl ceramide (the PK antigen) was 30-40% of normal. The ganglioside content of his erythrocytes was approximately four times normal and sialosylparagloboside was increased about sixfold. It appears that his erythrocytes was unable to synthesize normal quantities of trihexosyl ceramide, and that these serological and chemical features constitute a new phenotype in the P blood group system.

Adult↗
PubMed1976

Enzymatic synthesis of two fucose-containing glycolipids with fucosyltransferases of human serum.

Lacto-N-neotetraosylceramide incubated with human serum fucosyltransferase preparations gave rise to two fucoglycolipids. The faster migrating fucoglycolipid I on the basis of its thin-layer chromatographic mobility, susceptibility to alpha(1 leads to 2) fucosidase from Trichomonas foetus, radio-immunoprecipitation with Ulex europeus lectin and studies with Oh (Bombay) sera was identified as H-active glycolipid (H-I). The most probable structure of fucoglycolipid II should be that with fucose linked alpha(1 leads to 3) to N-acetylglucosamine. Lactosylceramide, ceramide trihexoside and globoside were not substrates for human serum fucosyltransferases. Lacto-N-neotetraosyl ceramide served as a fucose acceptor for all serum preparations tested while asialoganglioside was a substrate only when serum preparations containing H-gene dependent alpha-2-L-fucosyltransferase were used. With asialoganglioside only one radioactive reaction product was formed.

Blood Group Antigens↗
PubMed1986

Glycosphingolipid biosynthesis in early chick embryos.

Early chick embryos at the age of 22 h (neurula stage) and 48 h (20-25 somite stage) were explanted from eggs and cultured in vitro in the presence of radioactive sugar precursors. Metabolically labelled glycosphingolipids were isolated. Amongst these, neutral and acidic components, the latter including sulfatide and gangliosides, were identified. Cleavage by exoglycosidases, as well as immunostaining with antibodies on thin-layer plates, showed that at both embryonic stages glycosphingolipids were synthesized that belong to the globo series (globoside, Forssman glycolipid), the lacto series (lactoneotetraosylceramide, nLc4Cer, and two nLc4Cer-based gangliosides, a monosialo and a disialo species), and the ganglio series (ganglioside Gtet1a and higher sialylated derivatives).

Animals↗
PubMed1982

P-antigen-recognizing fimbriae from human uropathogenic Escherichia coli strains.

P-antigen-recognizing fimbriae (P fimbriae) from four pyelonephritogenic Escherichia coli strains and type 1 fimbriae from an E. coli strain and a Salmonella typhimurium strain were purified. The P fimbriae were morphologically similar to type 1 fimbriae. The purified P fimbriae agglutinated neuraminidase-treated human P1 and P2k erythrocytes but not p erythrocytes, which lack all P-blood group-specific glycosphingolipids. However, coating of neuraminidase-treated p erythrocytes with globoside rendered such erythrocytes agglutinable by the P fimbriae. The hemagglutinations were in all instances fully inhibited by the synthetic alpha-D-Galp-(1-4)-beta-D-Galp-1-O-Me glycoside. The binding specificity of the P fimbriae could also be demonstrated by using fimbriae coated onto latex particles and nontreated erythrocytes. It was thus concluded that the P fimbriae recognize and bind to the alpha-D-Galp-(1-4)-beta-D-Galp carbohydrate sequence occurring in the series of P-blood group antigen-specific glycosphingolipids. In contrast to both type 1 fimbriae, all four P fimbriae preparations showed multiple bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antisera were raised in rabbits against the various E. coli fimbriae. In enzyme-linked immunosorbent assays each one of the antisera to the P fimbriae reacted to titers of log 4 to 7 with both the homologous and the heterologous P fimbriae, but not with the type 1 fimbriae of E. coli. In a reciprocal fashion, the antiserum to the type 1 fimbriae of one E. coli strain reacted only with the homologous type 1 but not with any of the P fimbriae preparations.

Blood Group Antigens↗
PubMed1988

Detection of pilus subunits (pilins) and filaments by using anti-P pilin antisera.

P pilus filaments are important in binding to globoside through an adhesin located at the tip of the pilus. There is considerable antigenic variation among P pili, and the immunologic response is usually serotype specific. We purified denatured pilin subunits and used them as immunogens to prepare more broadly cross-reactive antisera. Although antifilament antisera (AFA) detected predominantly the homologous strain, antisubunit antisera (ASA) prepared from two different strains detected P pili in 16 of 16 and 14 of 16 P-piliated strains by Western blotting (immunoblotting). The binding of ASA to the homologous pilus filament was inhibited by only 3 of 17 strains. ASA agglutinated only two of nine heterologous strains and immunoprecipitated pili from one of three heterologous strains. By immunoelectron microscopy ASA was seen to bind to pilus filaments but not as strongly as AFA. Antiserum raised to the denatured pilin subunit was not substantially more reactive with pilus filaments derived from heterologous strains than was AFA. ASA was, however, a very useful probe for detecting most P pilins.

Agglutination Tests↗
PubMed1990

Binding of Pseudomonas aeruginosa to neutral glycosphingolipids of rabbit corneal epithelium.

35S-labeled Pseudomonas aeruginosa isolates were shown to bind to neutral glycosphingolipids (NGSLs) of rabbit corneal epithelia in culture by a thin-layer chromatogram overlay procedure. The lipids of the corneal epithelial cells grown in culture were extracted and partitioned into a chloroform-rich lower phase containing NGSLs and an aqueous upper phase containing gangliosides. By using a dot-blot assay, at least six times more radiolabeled P. aeruginosa isolates were shown to bind to the lipids in the lower phase compared with those in the upper phase. Thin-layer chromatography of the lower-phase lipids followed by staining with an orcinol spray revealed at least 10 NGSL components and several fast-migrating, nonglycosylated neutral lipid components (including cholesterol). 35S-labeled P. aeruginosa was shown to bind to NGSL components 1, 2, 5, 6, and 9. P. aeruginosa-reactive NGSL components 6 and 9 migrated with chromatographic mobilities similar to those of the standards ceramide trihexoside (CT) and ceramide monohexoside, respectively. Components 1 and 2 migrated slightly ahead of asialo GM1, and component 5 migrated faster than globoside but slower than CT. Among the various standards tested, P. aeruginosa bound to asialo GM1 and, to a lesser extent, to ceramide dihexoside and CT but not to GM1, GD1A, GM3, or ceramide monohexoside. It remains to be determined whether any of the five P. aeruginosa-reactive NGSL components of corneal epithelium identified in this study plays a role in the development of corneal infection. However, we have previously shown that component 9, one of the five P. aeruginosa-reactive NGSL components identified in this study, is present in significantly greater amounts in migrating epithelia than it is in nonmigrating epithelia (N. Panjwani, G. Michalopoulos, J. Song, G. Yogeeswaran, and J. Baum, Invest. Ophthalmol. Vis. Sci., in press). This may prove to be of biological significance because it is generally believed that traumatized (migrating) epithelia are more susceptible to infection than normal (nonmigrating) epithelia are.

Animals↗
PubMed1990

pap-2-encoded fimbriae adhere to the P blood group-related glycosphingolipid stage-specific embryonic antigen 4 in the human kidney.

A subtype of P fimbriae, encoded by the pap-2 gene cluster, has been analyzed for agglutination of erythrocytes and for binding to cryostat sections of the human kidney. We have demonstrated that pap-2-encoded fimbriae are capable of binding to erythrocytes from some animal species and to human erythrocytes which express globoside and the LKE (stage-specific embryonic antigen 4 [SSEA-4]) antigen. The pap-2 fimbriae bind to Bowman's capsule in the human kidney. Monoclonal antibodies directed against glycosphingolipids were used for the detection of specific P blood group-related antigens in the human kidney and on erythrocytes. Preincubation of kidney sections with monoclonal antibody MC813-70, which binds to the SSEA-4 antigen, inhibited adherence of purified pap-2-encoded fimbriae to Bowman's capsule. We suggest that one receptor for pap-2-encoded fimbriae is the antigen known as LKE (Luke) on human erythrocytes or SSEA-4 in the tissues.

Antibodies, Monoclonal↗
PubMed1993

The K88 fimbrial adhesin of enterotoxigenic Escherichia coli binds to beta 1-linked galactosyl residues in glycosphingolipids.

The K88 fimbrial adhesin enables certain strains of enterotoxigenic Escherichia coli to adhere to the porcine small intestine. In this study, the ability of the K88 adhesin to bind to glycosphingolipids was monitored by modified enzyme-linked immunosorbent assay and thin-layer chromatography overlay binding analysis. The binding of the K88 adhesin to glycosphingolipid-coated microtiter plates was saturable, with 50% maximal binding occurring with gangliotriaosylceramide, gangliotetraosylceramide, and lactosylceramide at 67 +/- 21, 117 +/- 21, and 73 +/- 22 pM, respectively. Thin-layer chromatography overlay binding analysis demonstrated that serotype O8:K87:K88ab:H19 E. coli bound to hydroxylated galactosylceramide, gangliotriaocylceramide, gangliotetraosylceramide, and lactosylceramide but not to globotriaosylceramide, nonhydroxylated galactosylceramide, glucosides, glucosylceramide, or a mixture of ceramides. The K88 adhesin did not bind by either assay to globoside, the Forssman glycolipid, GM1, GM2, GM3, GD1a, GD2, GD3, GQ1b, or GT1b. The binding pattern observed with the K88 adhesin suggests that beta 1-linked galactosyl residues are the minimum determinant required for binding, provided they are presented correctly. It is suggested that beta 1-linked galactose residues may form the molecular basis of both glycoprotein and glycolipid receptors for the K88 fimbrial adhesin in the porcine small intestine.

Adhesins, Escherichia coli↗
PubMed1994

Glycolipid receptors for attachment of Mycoplasma hyopneumoniae to porcine respiratory ciliated cells.

Glycolipid receptors for Mycoplasma hyopneumoniae attachment were analyzed by using a thin-layer chromatography (TLC) overlay assay. M. hyopneumoniae bound specifically to sulfatide, globoside, and monosialoganglioside GM3. No binding to sphingomyelin, cerebroside, lactosyl ceramide, ceramide trihexoside, monosialogangliosides GM1 and GM2, disialogangliosides (GD1a, GD1b, and GD3), trisialoganglioside (GT1b), cholesterol, cholesterol sulfate, palmitic acid, tripalmitin, or cholesteryl palmitate was detected. Total lipids extracted from cilia of the swine respiratory epithelium, the natural targets of M. hyopneumoniae infection, were also separated on TLC plates and overlaid with mycoplasmas. M. hyopneumoniae bound specifically to three ciliary glycolipids identified as La, Lb, and Lc. Binding to Lc was stronger than to La and Lb. All three lipids were believed to be sulfated glycolipids, as determined by laminin binding and staining with azure A. Lc was identified as a putative sulfatide because it has a mobility similar to that of authentic sulfatide and comigrated with sulfatide on TLC plates. Laminin bound to La, Lb, and Lc and produced dose-dependent inhibition of adherence of the mycoplasma to the three ciliary receptors. Binding of the mycoplasma to sulfatide, La, Lb, and Lc was partially inhibited by dextran sulfate, heparin, fucoidan, mucin, and chondroitin sulfate B. These substances blocked the adherence of M. hyopneumoniae to cilia and ciliated cells as shown in a previous study (Q. Zhang, T. F. Young, and R. F. Ross, Infect. Immun. 62:1616-1622, 1994). These results indicate that La, Lb, and Lc are the major native receptors for M. hyopneumoniae adherence to ciliated cells.

Animals↗
PubMed1996

Adherence to lipids and intestinal mucin by a recently recognized human pathogen, Campylobacter upsaliensis.

Campylobacter upsaliensis is a recently recognized human enteric pathogen associated with enteritis, colitis, bacteremia, and sepsis. Very little is known about the mechanisms of pathogenesis of this organism. The goals of this study were to determine whether C. upsaliensis binds to epithelial cells and whether there are specific lipid molecules that might serve as cell membrane receptors. In addition, we also explored C. upsaliensis binding to purified human small-intestinal mucin, since the mucus gel overlying the epithelium provides an initial contact surface for the bacteria and must be penetrated for the organisms to reach their cell receptors. Binding of C. upsaliensis to model epithelial cells was shown by microscopy adhesion assays, and binding to lipids was detected by thin-layer chromatography-overlay assays. Bacteria bound to phosphatidylethanolamine (PE), gangliotetraosylceramide (Gg4), and, more weakly, to phosphatidylserine (PS). There was no binding to ceramide, cholesterol, phosphatidylcholine, and globosides. Using receptor-based microtiter well immunoassays, we observed binding to be equal, specific, and saturable for PE and Gg 4 but low and nonspecific for PS. At least five bacterial surface proteins (50 to 90 kDa) capable of PE binding were identified by a lipid-silica affinity column technique. In slot blot overlay assays, biotin-labeled C. upsaliensis also bound in a concentration-dependent fashion to purified human small-intestinal mucin, implying that these microorganisms also express an adhesin(s) recognizing a specific mucin epitope(s). We speculate that binding to mucins may influence access of the bacteria to cell membrane receptors and thereby influence host resistance to infection.

Adhesins, Bacterial↗
PubMed1999

Binding of Actinobacillus pleuropneumoniae lipopolysaccharides to glycosphingolipids evaluated by thin-layer chromatography.

The binding profile of Actinobacillus pleuropneumoniae serotypes 1 and 2 to various glycosphingolipids was evaluated by using thin-layer chromatogram overlay. A. pleuropneumoniae whole cells recognized glucosylceramide (Glcbeta1Cer), galactosylceramide (Galbeta1Cer) with hydroxy and nonhydroxy fatty acids, sulfatide (SO(3)-3Galbeta1Cer), lactosylceramide (Galbeta1-4Glcbeta1Cer), gangliotriaosylceramide GgO3 (GalNAcbeta1-4Galbeta1-4Glcbeta1Cer), and gangliotetraosylceramide GgO4 (Galbeta1-3GalNAcbeta1-4Galbeta1-4Glcbeta1Cer) glycosphingolipids. We observed no binding to globoseries, globotriaosylceramide Gb3, globoside Gb4, or Forssman Gb5 glycosphingolipids or to gangliosides GM1, GM2, GM3, GD1a, GD1b, GD3, and GT1b. The A. pleuropneumoniae strains tested also failed to detect phosphatidylethanolamine or ceramide. Interestingly, extracted lipopolysaccharide (LPS) of serotype 1 and serotype 2 as well as detoxified LPS of serotype 1 showed binding patterns similar to that of whole bacterial cells. Binding to GlcCer, GalCer, sulfatide, and LacCer, but not to GgO3 and GgO4 glycosphingolipids, was inhibited after incubation of the bacteria with monoclonal antibodies against LPS O antigen. These findings indicate the involvement of LPS in recognition of three groups of glycosphingolipids: (i) GlcCer and LacCer, where glucose is probably an important saccharide sequence required for LPS binding; (ii) GalCer and sulfatide glycosphingolipids, where the sulfate group is part of the binding epitope of the isoreceptor; and (iii) GgO3 and GgO4, where GalNacbeta1-4Gal disaccharide represents the minimal common binding epitope. Taken together, our results indicate that A. pleuropneumoniae LPS recognize various saccharide sequences found in different glycosphingolipids, which probably represents a strong virulence attribute.

Actinobacillus pleuropneumoniae↗
PubMed1973

Tissue receptor for cholera exotoxin: postulated structure from studies with GM1 ganglioside and related glycolipids.

By a double-diffusion precipitation-in-gel technique, isolated cholera toxin as well as its natural toxoid were shown to be fixed and precipitated by the ganglioside G(M1) but not by any of the related glycolipids G(M3), G(M2), G(M1)-GlcNAc, G(D1a), G(D1b), G(T1), globoside, G(A1), and tetrahexoside-GlcNAc. Twenty-five nanograms of G(M1) was enough to give a precipitation line with 1.2 mug of toxin, whereas about 50 ng was required with this amount of toxoid. G(M1) also inactivated the toxin in the ileal loop as well as in the intradermal models in rabbits. A 1: 1 molar ratio of ganglioside to toxin was found limiting, e.g., 100 pg of G(M1) could inactivate 5 ng (about 50 blueing doses) of isolated toxin. G(M1) inactivated crude toxin (culture fil rate) with the same efficiency as isolated toxin, and the inactivating capacity of G(M1) was unaffected by mixing with other gangliosides, indicating the specificity in the reaction between G(M1) and toxin. The other glycolipids tested did not inactivate toxin except G(D1a) and G(A1) which did so with approximately 1,000 times less efficiency than G(M1). This identified the portion Gal --> GalNAc [Formula: see text] as the critical region in G(M1) for toxin fixation, and it is postulated that this may be the tissue receptor structure for the cholera toxin.

Animals↗
PubMed1973

Comparison of the tissue receptors for Vibrio cholerae and Escherichia coli enterotoxins by means of gangliosides and natural cholera toxoid.

The in vitro binding properties of enterotoxins of Vibrio cholerae and Escherichia coli to different pure gangliosides and related neutral glycosphin-golipids were analyzed with a sorbent assay utilizing plastic tubes to which the glycolipid substances had been coupled. It was found that the cholera toxin bound to G(M1) ganglioside better than to the other tested substances G(M3), G(M3)-NGN, G(M2), G(D1a), G(D1b), G(T), G(A1), tetrahexoside-GlcNac and globoside. With this assay using G(M1)-coated tubes it is possible to measure cholera toxin even at concentrations below 1 ng/ml. Also enterotoxin of various E. coli strains bound to G(M1), but the affinity was much less than for cholera toxin. The G(M1) ganglioside, in contrast to the other glycosphingolipids, effectively inactivated cholera toxin as determined with the intradermal and the ileal loop assays; approximately equimolar concentrations of the ganglioside in relation to toxin sufficed. Also, the skin and ileal loop activities of E. coli enterotoxins could be inhibited by G(M1); however, several orders more of the ganglioside were required for such inhibition than for inactivation of the cholera toxin, and the differences between G(M1) and the other substances were less pronounced for E. coli toxins. Preincubation of rabbit ileal loops with choleragenoid, a natural toxoid of V. cholerae which has binding properties to the G(M1) ganglioside similar to cholera toxin, made the loops resistant to subsequently added enterotoxin of V. cholerae. The responsiveness to enterotoxin of E. coli was not reduced by this toxoid. A likely interpretation of these data is that the G(M1) ganglioside constitutes or at least contains the structure of functional tissue receptors for the cholera toxin, whereas the weak binding to G(M1) by E. coli enterotoxins is probably a pathogenetically insignificant reflection of structural similarities between these toxins and cholera toxin. Consequently, the cholera toxoid by occupying functional intestinal G(M1) receptors for the cholera toxin could inhibit the ileal response to this toxin, but not the response to E. coli enterotoxin since the intestinal receptors for the latter toxin are not affected by the cholera toxoid.

Animals↗
PubMed1990

Rotaviruses specifically bind to the neutral glycosphingolipid asialo-GM1.

Rotaviruses are the major etiologic agents of severe diarrhea in children. Many rotaviruses encode a hemagglutinin which binds to sialic acids. We report that rotaviruses specifically recognize the neutral glycosphingolipid gangliotetraosylceramide (asialo-GM1 or GA1). GA1 resolved by thin-layer chromatography is bound by rotavirus, and binding is blocked by neutralizing rotavirus antiserum. Similar glycosphingolipid structures, such as globoside, gangliotriaosylceramide, and GA1 oxidized with galactose oxidase are ineffective in binding rotavirus. Other enteric viruses also specifically bind GA1. GA1 adsorbed to polystyrene beads inhibits rotavirus replication in vitro (as do anti-GA1 antibodies). The use of orally administered immobilized GA1 or anti-GA1 antibodies may prove useful in preventing or attenuating rotaviral and other enteric viral infections.

Animals↗
PubMed2001

Recombinant human parvovirus B19 vectors: erythrocyte P antigen is necessary but not sufficient for successful transduction of human hematopoietic cells.

The blood group P antigen, known to be abundantly expressed on erythroid cells, has been reported to be the cellular receptor for parvovirus B19. We have described the development of recombinant parvovirus B19 vectors with which high-efficiency, erythroid lineage-restricted transduction can be achieved (S. Ponnazhagan, K. A. Weigel, S. P. Raikwar, P. Mukherjee, M. C. Yoder, and A. Srivastava, J. Virol. 72:5224-5230, 1998). However, since a low-level transduction of nonerythroid cells could also be detected and since P antigen is expressed in nonerythroid cells, we reevaluated the role of P antigen in the viral binding and entry into cells. Cell surface expression analyses revealed that approximately 75% of primary human bone marrow mononuclear erythroid cells and approximately 31% of cells in the nonerythroid population were positive for P antigen. Two human erythroleukemia cell lines, HEL and K562, and a human promyelocytic leukemia cell line, HL-60, were also examined for P antigen expression and binding and entry of the vector. HEL and K562 cells showed intermediate levels, whereas HL-60 cells demonstrated high levels of expression of P antigen. However, the efficiency of vector binding to these cells did not correlate with P antigen expression. Moreover, despite P antigen positivity and efficient viral binding, HEL, K562, and HL-60 cells could not be transduced with the vector. Low levels of P antigen expression could also be detected in two primary cell types, human umbilical vein endothelial cells (HUVEC) and normal human lung fibroblasts (NHLF). In addition, vector binding occurred in both cell types and was inhibited by globoside, indicating the involvement of P antigen in virus binding to these cells. These primary cells could be efficiently transduced with the recombinant vector. These data suggest that (i) P antigen is expressed on a variety of cell types and is involved in binding of parvovirus B19 to human cells, (ii) the level of P antigen expression does not correlate with the efficiency of viral binding, (iii) P antigen is necessary but not sufficient for parvovirus B19 entry into cells, and (iv) parvovirus B19 vectors can be used to transduce HUVEC and NHLF. These studies further suggest the existence of a putative cellular coreceptor for efficient entry of parvovirus B19 into human cells.

Antigens, Surface↗