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• W2048651210 abstract "Sialylated glycans serve as cell surface attachment factors for a broad range of pathogens. We report an atypical example, where desialylation increases cell surface binding and infectivity of adeno-associated virus (AAV) serotype 9, a human parvovirus isolate. Enzymatic removal of sialic acid, but not heparan sulfate or chondroitin sulfate, increased AAV9 transduction regardless of cell type. Viral binding and transduction assays on mutant Chinese hamster ovary (CHO) cell lines defective in various stages of glycan chain synthesis revealed a potential role for core glycan residues under sialic acid in AAV9 transduction. Treatment with chemical inhibitors of glycosylation and competitive inhibition studies with different lectins suggest that N-linked glycans with terminal galactosyl residues facilitate cell surface binding and transduction by AAV9. In corollary, resialylation of galactosylated glycans on the sialic acid-deficient CHO Lec2 cell line with different sialyltransferases partially blocked AAV9 transduction. Quantitative analysis of AAV9 binding to parental, sialidase-treated or sialic acid-deficient mutant CHO cells revealed a 3–15-fold increase in relative binding potential of AAV9 particles upon desialylation. Finally, pretreatment of well differentiated human airway epithelial cultures and intranasal instillation of recombinant sialidase in murine airways enhanced transduction efficiency of AAV9 by >1 order of magnitude. Taken together, the studies described herein provide a molecular basis for low infectivity of AAV9 in vitro and a biochemical strategy to enhance gene transfer by AAV9 vectors in general. Sialylated glycans serve as cell surface attachment factors for a broad range of pathogens. We report an atypical example, where desialylation increases cell surface binding and infectivity of adeno-associated virus (AAV) serotype 9, a human parvovirus isolate. Enzymatic removal of sialic acid, but not heparan sulfate or chondroitin sulfate, increased AAV9 transduction regardless of cell type. Viral binding and transduction assays on mutant Chinese hamster ovary (CHO) cell lines defective in various stages of glycan chain synthesis revealed a potential role for core glycan residues under sialic acid in AAV9 transduction. Treatment with chemical inhibitors of glycosylation and competitive inhibition studies with different lectins suggest that N-linked glycans with terminal galactosyl residues facilitate cell surface binding and transduction by AAV9. In corollary, resialylation of galactosylated glycans on the sialic acid-deficient CHO Lec2 cell line with different sialyltransferases partially blocked AAV9 transduction. Quantitative analysis of AAV9 binding to parental, sialidase-treated or sialic acid-deficient mutant CHO cells revealed a 3–15-fold increase in relative binding potential of AAV9 particles upon desialylation. Finally, pretreatment of well differentiated human airway epithelial cultures and intranasal instillation of recombinant sialidase in murine airways enhanced transduction efficiency of AAV9 by >1 order of magnitude. Taken together, the studies described herein provide a molecular basis for low infectivity of AAV9 in vitro and a biochemical strategy to enhance gene transfer by AAV9 vectors in general. Cell surface glycans have been shown to play a critical role in the infectious pathways of viruses (1Olofsson S. Bergström T. Ann. Med. 2005; 37: 154-172Crossref PubMed Scopus (150) Google Scholar). Detailed studies of virus-glycan interactions have yielded significant insight into mechanisms underlying emergence and transmission of viral pathogens in different hosts. Among various glycolipids, glycoproteins, or proteoglycans anchored to the plasma membrane, sialylated glycans and heparan sulfate proteoglycans appear to serve as predominant substrates for viral attachment to the cell surface. For instance, heparan sulfate serves as a primary receptor for Herpesviridae (2Akhtar J. Shukla D. FEBS J. 2009; 276: 7228-7236Crossref PubMed Scopus (212) Google Scholar) as well as certain adenoviruses (3Dechecchi M.C. Melotti P. Bonizzato A. Santacatterina M. Chilosi M. Cabrini G. J. Virol. 2001; 75: 8772-8780Crossref PubMed Scopus (242) Google Scholar) and parvoviruses (4Summerford C. Samulski R.J. J. Virol. 1998; 72: 1438-1445Crossref PubMed Google Scholar, 5Schmidt M. Govindasamy L. Afione S. Kaludov N. Agbandje-McKenna M. Chiorini J.A. J. Virol. 2008; 82: 8911-8916Crossref PubMed Scopus (43) Google Scholar). Interactions between sialylated glycans and members of the Orthomyxoviridae (6Viswanathan K. Chandrasekaran A. Srinivasan A. Raman R. Sasisekharan V. Sasisekharan R. Glycoconj. J. 2010; 27: 561-570Crossref PubMed Scopus (84) Google Scholar), Reoviridae (7Guglielmi K.M. Johnson E.M. Stehle T. Dermody T.S. Curr. Top. Microbiol. Immunol. 2006; 309: 1-38PubMed Google Scholar), Polyomaviridae (8Neu U. Stehle T. Atwood W.J. Virology. 2009; 384: 389-399Crossref PubMed Scopus (81) Google Scholar) families and certain parvoviruses are also well known (9Nam H.J. Gurda-Whitaker B. Gan W.Y. Ilaria S. McKenna R. Mehta P. Alvarez R.A. Agbandje-McKenna M. J. Biol. Chem. 2006; 281: 25670-25677Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 10Walters R.W. Yi S.M. Keshavjee S. Brown K.E. Welsh M.J. Chiorini J.A. Zabner J. J. Biol. Chem. 2001; 276: 20610-20616Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 11Wu Z. Miller E. Agbandje-McKenna M. Samulski R.J. J. Virol. 2006; 80: 9093-9103Crossref PubMed Scopus (227) Google Scholar). Adeno-associated viruses (AAV) 2The abbreviations used are: AAV, adeno-associated virus; CBA, chicken β-actin; ConA, concanavalin A; ECL, Erythrina cristagalli lectin; HAE, human airway epithelia; MAL, Maackia amurensis lectin; m.o.i., multiplicity of infection; SNA lectin, Sambucus nigra lectin; UNC, University of North Carolina; vg, vector genomes; WGA, wheat germ agglutinin. are small, single-stranded DNA viruses that belong to the genus Dependovirus of the Parvoviridae family (12Bowles D.E. Rabinowitz J.E. Samulski R.J. Kerr J.R. Cotmore S.F. Bloom M.E. Parvoviruses. Edward Arnold Ltd., New York2006: 15-24Google Scholar). Recombinant AAV vectors, by virtue of their lack of pathogenicity and low immunogenicity, are currently being evaluated as lead candidates in clinical gene therapy trials (13Mueller C. Flotte T.R. Gene Ther. 2008; 15: 858-863Crossref PubMed Scopus (255) Google Scholar). The discovery of a large number of AAV isolates over the past decade has accelerated efforts to exploit tissue tropisms displayed by different strains for therapeutic gene transfer applications (14Gao G. Vandenberghe L.H. Wilson J.M. Curr. Gene Ther. 2005; 5: 285-297Crossref PubMed Scopus (413) Google Scholar, 15Mitchell A.M. Nicolson S.C. Warischalk J.K. Samulski R.J. Curr. Gene Ther. 2010; 10: 319-340Crossref PubMed Scopus (82) Google Scholar). Successful translation from bench to bedside will require a thorough understanding of molecular mechanisms underlying AAV infection. As with other viruses, attachment to cell surface glycans constitutes the first step in the AAV infectious pathway. For instance, several AAV serotypes have been shown to bind heparan sulfate proteoglycans (AAV2 (4Summerford C. Samulski R.J. J. Virol. 1998; 72: 1438-1445Crossref PubMed Google Scholar), AAV6 (11Wu Z. Miller E. Agbandje-McKenna M. Samulski R.J. J. Virol. 2006; 80: 9093-9103Crossref PubMed Scopus (227) Google Scholar)), whereas others utilize sialic acid for cell surface binding and entry (AAV4 (16Kaludov N. Brown K.E. Walters R.W. Zabner J. Chiorini J.A. J. Virol. 2001; 75: 6884-6893Crossref PubMed Scopus (336) Google Scholar), AAV5 (10Walters R.W. Yi S.M. Keshavjee S. Brown K.E. Welsh M.J. Chiorini J.A. Zabner J. J. Biol. Chem. 2001; 276: 20610-20616Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar), AAV1/6 (11Wu Z. Miller E. Agbandje-McKenna M. Samulski R.J. J. Virol. 2006; 80: 9093-9103Crossref PubMed Scopus (227) Google Scholar), bovine AAV (17Schmidt M. Chiorini J.A. J. Virol. 2006; 80: 5516-5522Crossref PubMed Scopus (33) Google Scholar)). Sialylated glycans that serve as primary receptors for the latter AAV strains vary at the level of N-acetylneuraminic acid linkage to underlying sugars, i.e. α2–3 or α2–6 linked to galactose residues (11Wu Z. Miller E. Agbandje-McKenna M. Samulski R.J. J. Virol. 2006; 80: 9093-9103Crossref PubMed Scopus (227) Google Scholar, 16Kaludov N. Brown K.E. Walters R.W. Zabner J. Chiorini J.A. J. Virol. 2001; 75: 6884-6893Crossref PubMed Scopus (336) Google Scholar). Further receptor specificity has been demonstrated at the level of N-linked or O-linked glycans displayed on the cell surface (11Wu Z. Miller E. Agbandje-McKenna M. Samulski R.J. J. Virol. 2006; 80: 9093-9103Crossref PubMed Scopus (227) Google Scholar, 16Kaludov N. Brown K.E. Walters R.W. Zabner J. Chiorini J.A. J. Virol. 2001; 75: 6884-6893Crossref PubMed Scopus (336) Google Scholar). Selective recognition of such linkages and underlying core glycan types (18Cohen M. Varki A. OMICS. 2010; 14: 455-464Crossref PubMed Scopus (168) Google Scholar) is likely enabled by differences in the capsid surface topology of AAV serotypes (19Govindasamy L. Padron E. McKenna R. Muzyczka N. Kaludov N. Chiorini J.A. Agbandje-McKenna M. J. Virol. 2006; 80: 11556-11570Crossref PubMed Scopus (137) Google Scholar, 20Ng R. Govindasamy L. Gurda B.L. McKenna R. Kozyreva O.G. Samulski R.J. Parent K.N. Baker T.S. Agbandje-McKenna M. J. Virol. 2010; 84: 12945-12957Crossref PubMed Scopus (106) Google Scholar). In general, dependence of AAV infectivity on sialic acid has been demonstrated using a battery of chemical, biochemical, and genetic tools to desialylate cell surface glycans. The current study is focused on glycan interactions of a human AAV isolate, AAV serotype 9/Hu.14 (Clade F) (21Gao G. Vandenberghe L.H. Alvira M.R. Lu Y. Calcedo R. Zhou X. Wilson J.M. J. Virol. 2004; 78: 6381-6388Crossref PubMed Scopus (762) Google Scholar). Recombinant AAV9 vectors display widespread and robust transduction following systemic administration in animal models but fail to infect cells in culture (21Gao G. Vandenberghe L.H. Alvira M.R. Lu Y. Calcedo R. Zhou X. Wilson J.M. J. Virol. 2004; 78: 6381-6388Crossref PubMed Scopus (762) Google Scholar, 22Zincarelli C. Soltys S. Rengo G. Rabinowitz J.E. Mol. Ther. 2008; 16: 1073-1080Abstract Full Text Full Text PDF PubMed Scopus (898) Google Scholar). We demonstrate that efficient gene transfer by AAV9 vectors requires an atypical interaction with nonsialylated cell surface glycans. The results described herein provide a molecular basis for the low infectivity of AAV9 observed in cell culture. All plasmids were obtained from the University of North Carolina (UNC) vector core. The triple plasmid transfection protocol (23Grieger J.C. Choi V.W. Samulski R.J. Nat. Protoc. 2006; 1: 1412-1428Crossref PubMed Scopus (400) Google Scholar) utilized for production of AAV9 vectors includes (i) the AAV helper plasmid, pXR9, containing AAV2 Rep and AAV9 Cap genes; (ii) the adenoviral helper plasmid, pXX6-80; and (iii) the vector genome cassette, pTR-CBA-Luc or pHpa-trs-SK, containing the firefly luciferase gene driven by the chicken β-actin (CBA) promoter or self-complementary GFP cassette driven by the cytomegalovirus (CMV) promoter, respectively. The vector genome cassette is flanked by inverted terminal repeats required for packaging. The inverted terminal repeats are the only elements within the vector genome cassette derived from the wild-type AAV genome, thereby eliminating 96% of viral elements. Recombinant AAV9 vectors generated thus allow quantitation of viral infectivity (or transduction efficiency) through luciferase transgene expression assays. HEK293 cells utilized for production of recombinant AAV9 vectors were obtained from the UNC vector core. Sonicated cell lysates and PEG8000 precipitates from supernatant were pooled and subjected to cesium chloride ultracentrifugation as described earlier (23Grieger J.C. Choi V.W. Samulski R.J. Nat. Protoc. 2006; 1: 1412-1428Crossref PubMed Scopus (400) Google Scholar). Dialyzed peak fractions were subjected to quantitative PCR using a Roche Light Cycler instrument with luc transgene-specific primers to determine viral vector titers (forward, 5′-AAA AGC ACT CTG ATT GAC AAA TAC-3′; reverse, 5′-CCT TCG CTT CAA AAA ATG GAA C-3′). All cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin, streptomycin, amphotericin B (Sigma) and maintained in 5% CO2 at 37 °C unless mentioned otherwise. COS-1 (monkey kidney), Neuro2a (mouse neuroblastoma), U87 (human glioma) and Huh7 (human hepatocarcinoma) cells were obtained from the UNC tissue culture facility and utilized in viral transduction assays. Chinese hamster ovary (CHO) Pro5 and mutant Lec1, Lec2 cell lines were a gift from Dr. Jude Samulski (UNC Chapel Hill), and the CHO Lec8 cell line was purchased from American Type Culture Collection. All CHO cells, utilized for viral binding and transduction assays, were cultured in α-MEM (GIBCO) supplemented with 10% FBS and penicillin, streptomycin, and amphotericin B as outlined above. Well differentiated human airway epithelial (HAE) cultures (4–6 weeks) grown on permeable membrane supports (Millipore, Corning, NY) at the air-liquid interface were provided by the Cell Culture Models Core and the UNC Cystic Fibrosis/Pulmonary Research Center. Different cell lines were seeded at 105 cells/well in 24-well plates and allowed to adhere overnight at 37 °C. Plates were then prechilled at 4 °C for 30 min and incubated with AAV9 vectors at a multiplicity of infection (m.o.i.) of 1000 vector genomes (vg)/cell to allow binding to the cell surface for 1.5 h at 4 °C. Unbound virions were then removed by washing three times with ice-cold 1× phosphate-buffered saline (1× PBS), and 0.5 ml of DMEM added to each well. Luciferase transgene expression levels were quantitated after incubation for 24 h from cell lysates using a Victor 2 luminometer (PerkinElmer Life Sciences). For studies with HAE, well differentiated cultures were pretreated with 1.25 units/ml sialidase A (Prozyme GK80040) at 37 °C for 3 h followed by three washes with ice-cold 1× PBS. Cultures were then incubated with scAAV9-CMV-GFP vectors (m.o.i. = 105 vg/cell) as outlined above. Fluorescence micrographs of green fluorescent protein (GFP) expression in HAE cultures at 2 weeks after transduction were obtained using an Olympus epifluorescence microscope equipped with a 20× objective and a Hamamatsu camera. Different cell lines were treated with heparinase I and III (from Flavobacterium heparinum; Sigma H2519 and H8891), chondroitinase ABC (from Proteus vulgaris; Sigma C2905), and neuraminidase type III (from Vibrio cholerae; Sigma N7885) to determine the role of cell surface glycans in AAV9 infection. Briefly, COS-1 cells were seeded at 105 cells/well in 24-well plates and pretreated with 50 milliunits/ml neuraminidase, 3 units/ml heparinase I, 1.5 units/ml heparinase III, and 1.5 units/ml chondroitinase ABC in DMEM at 37 °C for 2 h. Neuro2a, U87, HEK293, and Huh7 cells were treated with neuraminidase alone. Cells were then washed three times with 1× PBS and subjected to AAV9 infection at an m.o.i. of 1000 vg/cell. Luciferase transgene expression assays were carried out as described earlier at 24 h after infection. CHO Lec2 cells were seeded at 105 cells/well in 24-well plates and pretreated for 24 h with small molecule inhibitors of glycosylation, Swainsonine (10 μm; Sigma S8195) and α-benzyl-GalNAc (1 μg/ml; Sigma B4894) to determine the role of N- and O-glycans in AAV9 infection. Cells pretreated with chemicals were subjected to AAV9 infection at an m.o.i. of 1000 vg/cell and luciferase transgene expression assays carried out as described earlier. The sialic acid-deficient cell line, CHO Lec2 was treated with 50 milliunits/ml each of α2,3-(N)-sialyltransferase (Calbiochem 566218), α2,6-(N)-sialyltransferase (Calbiochem 566222), or α2,3-(O)-sialyltransferase (Calbiochem 566227) and 1 mm CMP-sialic acid (Sigma) in medium for 3 h at 37 °C. Untreated CHO Pro5 cells with endogenous sialic acid were included as control. Cells were then rinsed three times with 1× PBS and subjected to AAV9 infection at an m.o.i. of 1000 vg/cell and luciferase transgene expression assays carried out as described earlier. Competitive inhibition of AAV9 infection was carried out using a panel of lectins, concanavalin A (ConA), wheat germ agglutinin (WGA), Maackia amurensis lectin (MAL I), Sambucus nigra lectin (SNA), and Erythrina cristagalli lectin (ECL) obtained from Vector Laboratories (Burlingame, CA). Briefly, prechilled CHO Pro5 or mutant Lec2 cells were incubated with 100 μg/ml FITC-conjugated lectin (fluorescent labeling assay) in α-MEM or along with AAV9 particles (transduction assay at m.o.i. = 10,000 vg/cell) for a period 1.5 h at 4 °C. After three washes with ice-cold 1× PBS to remove unbound virions and/or lectins, cells were imaged using an Olympus epifluorescence microscope equipped with a Hamamatsu camera or incubated for 24 h at 37 °C prior to luciferase transgene expression analysis. CHO Pro5 cells were seeded at a density of 104 cells/well in 96-well plates prior to treatment with 50 milliunits/ml neuraminidase type III from V. cholerae for 2 h at 37 °C. Untreated Pro5 and Lec2 cells were included as negative and positive controls, respectively. Cells were then prechilled at 4 °C for 30 min, followed by incubation with AAV9 particles at m.o.i. of 102, 5 × 102, 103, 5 × 103, 104, 5 × 104, 105, 5 × 105 vg/cell for 1.5 h at 4 °C. Cells were then subjected to three washes with ice-cold 1× PBS to remove unbound virions. Cell surface-bound virions were collected along with cell lysates following three freeze-thaw cycles and vg copy numbers/cell determined using quantitative PCR as outlined earlier. Binding curves were generated using GraphPad Prism 5 software by applying the single site binding model (Y = Bmax*X/(Kd′ + X)), where Y represents the number of bound virions/cell determined by quantitative PCR; X represents m.o.i.; Bmax is the maximum binding capacity, and Kd′, the observed disassociation constant. All experiments were carried out with 6–8-week old female BALB/c mice (Jackson Laboratories, Bar Harbor, ME) maintained and treated in accordance with National Institutes of Health guidelines and as approved by IACUC at UNC-Chapel Hill. Mice were administered via intranasal instillation with either 100 μl of PBS (50 μl/nostril) or 100 μl of neuraminidase type III from V. cholerae (200 milliunits; Sigma). At 2 h after treatment, a dose of 5 × 1010 AAV9 particles in 1× PBS (50 μl/nostril) was administered. Luciferase transgene expression in live animals was obtained using a Xenogen IVIS Lumina® imaging system (Caliper Lifesciences, CA) after intranasal instillation of luciferin substrate (120 mg/kg; Nanolight). Image analysis was carried out using the Living Image software® (Caliper Lifesciences) and luciferase expression reported in relative light units (photons/s per cm2 per steradian). Enzymatic removal of different cell surface glycans was achieved by treating cells with different glycosidases. Removal of terminal sialic acid residues using neuraminidase (from V. cholerae) enhanced AAV9 transduction by >1 log unit compared with virus alone on COS-1 cells (Fig. 1A). In contrast, hydrolysis of cell surface heparan sulfate or chondroitin sulfate proteoglycans using heparinase I/III or chondroitinase ABC, respectively, had no effect on viral transduction compared with control. Neuraminidase treatment abrogated AAV1 transduction, whereas AAV2 remained unaffected under these conditions (Fig. 1B). In addition, treatment of cell lines derived from different tissues with neuraminidase (gray bars) prior to AAV9 infection enhanced transduction efficiency by nearly 2 log units in contrast to infection with virus alone (white bars) (Fig. 1C). These results were corroborated by increased binding and internalization of AAV9, but not AAV1 vectors upon sialidase treatment in these cell lines (supplemental Figs. S1 and S2). Thus, sialic acid appears to mask cell surface glycans that selectively facilitate AAV9 infection in vitro. Further, enzymatic desialylation might serve as a facile biochemical strategy to enhance transduction efficiency of AAV9 vectors in vitro and might enable detailed analysis of intracellular trafficking pathways. Analysis of cell surface binding and infectivity of AAV1 and AAV9 on parental (Pro5) and mutant CHO cell lines was carried out to further understand the role of core glycans under sialic acid in AAV9 infection. The CHO Lec2 cell line lacks terminal sialic acid due to a defect in CMP-sialic acid transport (24Deutscher S.L. Nuwayhid N. Stanley P. Briles E.I. Hirschberg C.B. Cell. 1984; 39: 295-299Abstract Full Text PDF PubMed Scopus (198) Google Scholar), whereas Lec8 and Lec1 cell lines are defective in translocation of UDP-galactose and N-acetylglucosaminyl transferase activity (25Deutscher S.L. Hirschberg C.B. J. Biol. Chem. 1986; 261: 96-100Abstract Full Text PDF PubMed Google Scholar, 26Stanley P. Chaney W. Mol. Cell. Biol. 1985; 5: 1204-1211Crossref PubMed Scopus (32) Google Scholar), respectively. Correspondingly, cell surface glycans on CHO Lec2 cells contain terminal galactosyl residues, whereas Lec8 and Lec1 cell lines display predominantly terminal N-acetylglucosamine and mannosylated glycans, respectively (Fig. 2A). As seen in Fig. 2, B and C, cell surface binding and transduction of AAV9 particles (gray bars) on Lec2 cells are significantly increased (>1 log unit) compared with the parental Pro5 cell line. In contrast, AAV1 (white bars), which requires sialic acid for infection, shows ∼10-fold decrease in binding and transduction in all CHO Lec mutant cell lines. In addition, no major changes in binding and infectivity are observed in case of Lec8 and Lec1 cells for AAV9 particles. These results suggest that galactosylated glycans immediately underlying sialic acid can facilitate AAV9 cell surface binding and entry. The results also support the notion that AAV9 particles might exploit an inefficient and nonspecific uptake mechanism in the parental Pro5 and mutant Lec8 and Lec1 cell lines. To elucidate the nature of glycans required for AAV9 infection further, we utilized small molecule inhibitors of glycosylation and sialyltransferases to modify terminal galactosyl residues on the sialic acid-deficient Lec2 cell surface. Swainsonine (27Elbein A.D. Solf R. Dorling P.R. Vosbeck K. Proc. Natl. Acad. Sci. U.S.A. 1981; 78: 7393-7397Crossref PubMed Scopus (225) Google Scholar) and α-benzyl-O-GalNAc (28Kuan S.F. Byrd J.C. Basbaum C. Kim Y.S. J. Biol. Chem. 1989; 264: 19271-19277Abstract Full Text PDF PubMed Google Scholar) are chemical inhibitors of N-linked and O-linked glycosylation, respectively. Treatment with these reagents results in a corresponding decrease in cell surface expression of N-linked glycans and O-linked glycans. As seen in Fig. 3A, AAV9 infection is significantly blocked by Swainsonine (∼ 75%), whereas α-benzyl-O-GalNAc has a modest inhibitory effect (∼25%). These results suggest that AAV9 prefers N-linked cell surface glycans for infection. In addition, resialylation of the sialic acid-deficient Lec2 cell surface was carried out to understand the nature of the sialic acid linkage that blocks AAV9 infection (Fig. 3B). The abilities of different sialyltransferases to partially block AAV9 infection was observed to follow the order α2,3-N-sialyltransferase (3′NST) > α2,6-N-sialyltransferase (6′NST) > α2,3-O-sialyltransferase (3′OST). Alteration of cell surface glycans upon resialylation was confirmed by staining with lectins recognizing different glycan residues and linkages (Fig. 3C). As expected Pro5 cells expressing sialylated glycans and Lec2 cells expressing asialoglycans demonstrate preferential staining by FITC-MAL I and FITC-ECL, respectively. Resialylation of Gal(β1,4)GlcNAc residues with 3′NST, but not 3′OST partially restores FITC-MAL-I staining concurrent with decrease in AAV9 transduction. The lack of MAL I staining in 6′NST-treated Lec2 cells is expected due to lack of recognition of α2,6-sialylated glycans by MAL I. Taken together, these results not only corroborate the important role played by core N-linked glycans in AAV9 infection, but also the potential for α2,3 and α2,6 sialic acid linkages to block infection by masking underlying glycoconjugates on the cell surface. To understand better the nature of AAV9-glycan interactions that mediate infection, we carried out competitive inhibition studies with lectins that recognize different glycan linkages on the cell surface (Fig. 4, A and B). Specifically, we utilized (i) MAL I, which recognizes native, α2,3-sialylated, or sulfated glycoconjugates having Gal(β1,4)N-GlcNAc structures (29Wang W.C. Cummings R.D. J. Biol. Chem. 1988; 263: 4576-4585Abstract Full Text PDF PubMed Google Scholar, 30Bai X. Brown J.R. Varki A. Esko J.D. Glycobiology. 2001; 11: 621-632Crossref PubMed Scopus (45) Google Scholar); (ii) SNA lectin, which binds preferentially to α2,6-sialylated galactose residues (31Shibuya N. Goldstein I.J. Broekaert W.F. Nsimba-Lubaki M. Peeters B. Peumans W.J. J. Biol. Chem. 1987; 262: 1596-1601Abstract Full Text PDF PubMed Google Scholar); (iii) ECL, which demonstrates specificity toward galactose residues, in particular, Gal(β1,4)N-GlcNAc (32Wu A.M. Wu J.H. Tsai M.S. Yang Z. Sharon N. Herp A. Glycoconj. J. 2007; 24: 591-604Crossref PubMed Scopus (41) Google Scholar); (iv) WGA, which binds N-GlcNAc and tolerates glycoconjugates containing sialic acid with different linkages (33Yamamoto K. Tsuji T. Matsumoto I. Osawa T. Biochemistry. 1981; 20: 5894-5899Crossref PubMed Scopus (186) Google Scholar); and (v) ConA, which recognizes mannose residues (34Ohyama Y. Kasai K. Nomoto H. Inoue Y. J. Biol. Chem. 1985; 260: 6882-6887Abstract Full Text PDF PubMed Google Scholar). The SNA lectin had no effect on AAV9 infection and can be explained by low levels of α2,6-sialylated glycans in both cell lines of rodent (hamster) origin (35Stults N.L. Fechheimer M. Cummings R.D. J. Biol. Chem. 1989; 264: 19956-19966Abstract Full Text PDF PubMed Google Scholar). On the other hand, the MAL I demonstrated 5–10-fold inhibition of AAV9 infection in both Pro5 and Lec2 cell lines. More importantly, a striking difference in AAV9 transduction was observed in case of ECL-treated cells with 5-fold inhibition in parental Pro5 cells and nearly 200-fold inhibitory activity in the Lec2 cell line, demonstrating the importance of core Galβ1-linked residues. The ConA and WGA lectins demonstrated broad inhibitory activity in both parental Pro5 cells and the sialic acid-deficient Lec2 cell line suggesting that underlying N-GlcNAc and core mannose residues might contribute to AAV9-glycan interactions. These results were corroborated by fluorescent staining of Pro5 and Lec2 cell lines with aforementioned lectins (Fig. 4C). As expected, MAL I and WGA lectin, which bind to α2,3-sialylated glycans, preferentially label Pro5 cells. In contrast, preferential ECL staining is noted in Lec2 cells. The SNA lectin, which recognizes α2,6-sialic acid, does not stain either Pro5 or Lec2 cells, confirming the lack (or modest expression) of α2,6-sialylated glycans on these hamster-derived cell lines. Further evidence supporting the important role of galactose in mediating AAV9 infection was obtained by treatment of CHO Pro5 cells with endo-β-galactosidase (supplemental Fig. S3, A and B). Removal of galactose residues, but not fucose abrogated AAV9 transduction. Taken together, these results suggest that terminal galactosyl residues serve as the primary receptor for AAV9. Cell surface binding curves were generated to establish a quantitative biochemical rationale for the observed increase in AAV9 transduction following desialylation. Binding of AAV9 particles to the surface of parental CHO Pro5 cells, sialidase-treated Pro5 cells, or sialic acid-deficient Lec2 cells was carried out over a range of multiplicities of infection (vg/cell). As seen in Fig. 5, enzymatic removal of sialic acid partially recapitulates the effect of genetic desialylation by increasing the number of cell surface-bound AAV9 particles. Nonlinear regression analyses of binding data were carried out using the single site binding model (Y = Bmax*X/(Kd′ + X)), where X and Y represent multiplicity of infection and number of bound AAV9 particles, respectively; Bmax is the maximum binding capacity, and Kd′ is the relative (observed) binding affinity. Calculation of aforementioned parameters reveals Bmax values averaging 375 vg/cell on wild-type Pro5 cells, a ∼3-fold increase to 1025 vg/cell on sialidase-treated Pro5 cells, and a ∼5-fold increase to 1971 vg/cell on the sialic acid-deficient Lec2 cell line. These results support the observation that AAV9 prefers asialo N-glycans with terminal Galβ1-linked residues. In addition, an apparent decrease in Kd′ (∼3-fold) is observed in Lec2 cells as well as a corresponding 15-fold increase in relative binding potential (Bmax/Kd′) (Table 1). These results support the notion that enhanced avidity plays a role in mediating AAV9 binding to asialo N-glycans.TABLE 1Binding parameters for AAV9 interactions with cell surface glycansParametersPro5Pro5 + SialidaseLec2Bmax (vg/cell)3.75 × 102 ± 0.86 × 1021.03 × 103 ± 0.19 × 1031.97 × 103 ± 0.25 × 103Kd′ (vg/cell)3.58 × 105 ± 1.64 × 1053.34 × 105 ± 1.26 × 1051.22 × 105 ± 0.41 × 105Bmax/Kd′1.05 × 10−33.07 × 10−31.62 × 10−2R20.970.960.96 Open table in a new tab To evaluate the potential of enzymatic desialylation as a strategy to enhance gene transfer by AAV9 vectors, we evaluated the effe" @default.
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