FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1970604893> ?p ?o ?g. }

• W1970604893 endingPage "40692" @default.
• W1970604893 startingPage "40681" @default.
• W1970604893 abstract "The recognition of influenza A virus (IAV) by surfactant protein D (SP-D) is mediated by interactions between the SP-D carbohydrate recognition domains (CRD) and glycans displayed on envelope glycoproteins. Although native human SP-D shows potent antiviral and aggregating activity, trimeric recombinant neck+CRDs (NCRDs) show little or no capacity to influence IAV infection. A mutant trimeric NCRD, D325A/R343V, showed marked hemagglutination inhibition and viral neutralization, with viral aggregation and aggregation-dependent viral uptake by neutrophils. D325A/R343V exhibited glucose-sensitive binding to Phil82 hemagglutinin trimer (HA) by surface plasmon resonance. By contrast, there was very low binding to the HA trimer from another virus (PR8) that lacks glycans on the HA head. Mass spectrometry demonstrated the presence of high mannose glycans on the Phil82 HA at positions known to contribute to IAV binding. Molecular modeling predicted an enhanced capacity for bridging interactions between HA glycans and D325A/R343V. Finally, the trimeric D325A/R343V NCRD decreased morbidity and increased viral clearance in a murine model of IAV infection using a reassortant A/WSN/33 virus with a more heavily glycosylated HA. The combined data support a model in which altered binding by a truncated mutant SP-D to IAV HA glycans facilitates viral aggregation, leading to significant viral neutralization in vitro and in vivo. These studies demonstrate the potential utility of homology modeling and protein structure analysis for engineering effective collectin antivirals as in vivo therapeutics. The recognition of influenza A virus (IAV) by surfactant protein D (SP-D) is mediated by interactions between the SP-D carbohydrate recognition domains (CRD) and glycans displayed on envelope glycoproteins. Although native human SP-D shows potent antiviral and aggregating activity, trimeric recombinant neck+CRDs (NCRDs) show little or no capacity to influence IAV infection. A mutant trimeric NCRD, D325A/R343V, showed marked hemagglutination inhibition and viral neutralization, with viral aggregation and aggregation-dependent viral uptake by neutrophils. D325A/R343V exhibited glucose-sensitive binding to Phil82 hemagglutinin trimer (HA) by surface plasmon resonance. By contrast, there was very low binding to the HA trimer from another virus (PR8) that lacks glycans on the HA head. Mass spectrometry demonstrated the presence of high mannose glycans on the Phil82 HA at positions known to contribute to IAV binding. Molecular modeling predicted an enhanced capacity for bridging interactions between HA glycans and D325A/R343V. Finally, the trimeric D325A/R343V NCRD decreased morbidity and increased viral clearance in a murine model of IAV infection using a reassortant A/WSN/33 virus with a more heavily glycosylated HA. The combined data support a model in which altered binding by a truncated mutant SP-D to IAV HA glycans facilitates viral aggregation, leading to significant viral neutralization in vitro and in vivo. These studies demonstrate the potential utility of homology modeling and protein structure analysis for engineering effective collectin antivirals as in vivo therapeutics. Surfactant Protein D (SP-D) 2The abbreviations used are: SP-Dsurfactant protein DCRDcarbohydrate recognition domainIAVinfluenza A virusNCRDneck+carbohydrate recognition domainhNCRDhuman NCRDSPRsurface plasmon resonanceMBPmannose-binding proteinHAIhemagglutination inhibitionNi-NTAnickel-nitrilotriacetic acidHBSHepes-buffered saline. is a member of a family of collagenous C-type lectins, or collectins. This family includes mannose-binding protein (MBP), surfactant protein A, and certain bovine serum homologs, which are evolutionary descendents of SP-D. All collectins are characterized by an N-terminal collagen domain and a C-terminal lectin domain, and all appear to have roles in innate immunity. surfactant protein D carbohydrate recognition domain influenza A virus neck+carbohydrate recognition domain human NCRD surface plasmon resonance mannose-binding protein hemagglutination inhibition nickel-nitrilotriacetic acid Hepes-buffered saline. There is substantial evidence that SP-D contributes to the neutralization and clearance of respiratory viruses (1Crouch E.C. Laurent G. Shapiro S. Encyclopedia of Respiratory Medicine. 4. Elsevier Ltd., Oxford, UK2006: 152-158Crossref Scopus (3) Google Scholar, 2Kishore U. Greenhough T.J. Waters P. Shrive A.K. Ghai R. Kamran M.F. Bernal A.L. Reid K.B. Madan T. Chakraborty T. Mol. Immunol. 2006; 43: 1293-1315Crossref PubMed Scopus (438) Google Scholar, 3Wright J.R. Nat. Rev. Immunol. 2005; 5: 58-68Crossref PubMed Scopus (786) Google Scholar, 4Whitsett J.A. Biol. Neonate. 2005; 88: 175-180Crossref PubMed Scopus (56) Google Scholar, 5Holmskov U. Thiel S. Jensenius J.C. Annu. Rev. Immunol. 2003; 21: 547-578Crossref PubMed Scopus (660) Google Scholar). For example, SP-D-deficient mice show delayed clearance and heightened inflammatory responses to strains of respiratory syncytial virus and influenza A virus (IAV) (6LeVine A.M. Whitsett J.A. Hartshorn K.L. Crouch E.C. Korfhagen T.R. J. Immunol. 2001; 167: 5868-5873Crossref PubMed Scopus (244) Google Scholar, 7LeVine A.M. Elliott J. Whitsett J. Srikiatkhachorn A. Crouch E. DeSilva N. Korfhagen T. Am. J. Respir. Crit. Care Med. 2004; 31: 193-199Google Scholar, 8Vigerust D.J. Ulett K.B. Boyd K.L. Madsen J. Hawgood S. McCullers J.A. J. Virol. 2007; 81: 8593-8600Crossref PubMed Scopus (150) Google Scholar, 9Hawgood S. Brown C. Edmondson J. Stumbaugh A. Allen L. Goerke J. Clark H. Poulain F. J. Virol. 2004; 78: 8565-8572Crossref PubMed Scopus (101) Google Scholar). Significantly, SP-D-deficient mice show a selective impairment in clearance of strains reactive with SP-D in vitro (6LeVine A.M. Whitsett J.A. Hartshorn K.L. Crouch E.C. Korfhagen T.R. J. Immunol. 2001; 167: 5868-5873Crossref PubMed Scopus (244) Google Scholar, 9Hawgood S. Brown C. Edmondson J. Stumbaugh A. Allen L. Goerke J. Clark H. Poulain F. J. Virol. 2004; 78: 8565-8572Crossref PubMed Scopus (101) Google Scholar) and can be rescued by transgenic overexpression of trimeric rat SP-D subunits or with purified native human SP-D (6LeVine A.M. Whitsett J.A. Hartshorn K.L. Crouch E.C. Korfhagen T.R. J. Immunol. 2001; 167: 5868-5873Crossref PubMed Scopus (244) Google Scholar, 10Zhang L. Hartshorn K.L. Crouch E.C. Ikegami M. Whitsett J.A. J. Biol. Chem. 2002; 277: 22453-22459Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Binding of SP-D to IAV involves interactions with asparagine-linked glycans located near the sialic acid receptor on the hemagglutinin (11Hartshorn K.L. White M.R. Voelker D. R Coburn J. Zaner K. Crouch E.C. Biochem. J. 2000; 351: 449-458Crossref PubMed Scopus (123) Google Scholar, 12Reading P.C. Holmskov U. Anders E.M. J. Gen. Virol. 1998; 79: 2255-2263Crossref PubMed Scopus (48) Google Scholar, 13Hartshorn K.L. Webby R. White M.R. Tecle T. Pan C. Boucher S. Moreland R.J. Crouch E.C. Scheule R.K. Respir. Res. 2008; 9: 65Crossref PubMed Scopus (84) Google Scholar). Furthermore, endoglycosidase H eliminates binding of SP-D to denatured HA in viral lysates, indicating preferential interactions with high mannose or hybrid oligosaccharides (11Hartshorn K.L. White M.R. Voelker D. R Coburn J. Zaner K. Crouch E.C. Biochem. J. 2000; 351: 449-458Crossref PubMed Scopus (123) Google Scholar). Viral interactions require calcium and are specifically inhibited by monosaccharide ligands of SP-D, directly implicating the lectin activity of the SP-D carbohydrate recognition domain (CRD) (11Hartshorn K.L. White M.R. Voelker D. R Coburn J. Zaner K. Crouch E.C. Biochem. J. 2000; 351: 449-458Crossref PubMed Scopus (123) Google Scholar). This is consistent with crystallographic studies of recombinant trimeric neck plus carbohydrate recognition domains (NCRDs) that have demonstrated binding of the equatorial hydroxyl groups of mannoses to calcium at the lectin site (14Crouch E. McDonald B. Smith K. Roberts M. Mealy T. Seaton B. Head J. Biochemistry. 2007; 46: 5160-5169Crossref PubMed Scopus (28) Google Scholar, 15Wang H. Head J. Kosma P. Brade H. Müller-Loennies S. Sheikh S. McDonald B. Smith K. Cafarella T. Seaton B. Crouch E. Biochemistry. 2008; 47: 710-720Crossref PubMed Scopus (54) Google Scholar, 16Shrive A.K. Tharia H.A. Strong P. Kishore U. Burns I. Rizkallah P.J. Reid K.B. Greenhough T.J. J. Mol. Biol. 2003; 331: 509-523Crossref PubMed Scopus (97) Google Scholar). SP-D and the native serum collectins differ in their capacity to interact with respiratory viruses. For example, native bovine serum conglutinin and bovine serum CL-43 show greater binding to IAV than SP-D (17Hartshorn K.L. Holmskov U. Hansen S. Zhang P. Meschi J. Mogues T. White M.R. Crouch E.C. Biochem. J. 2002; 366: 87-96Crossref PubMed Google Scholar, 18Hartshorn K.L. Sastry K. Brown D. White M.R. Okarma T.B. Lee Y.M. Tauber A.I. J. Immunol. 1993; 151: 6265-6273PubMed Google Scholar, 19Hartshorn K.L. Sastry K. White M.R. Anders E.M. Super M. Ezekowitz R.A. Tauber A.I. J. Clin. Invest. 1993; 91: 1414-1420Crossref PubMed Scopus (183) Google Scholar). Alignments of these collectins have revealed non-conservative substitutions at positions corresponding to 325 and 343 (Table 1) that are highly exposed on the opposing N-terminal and C-terminal ridges of the carbohydrate binding groove, respectively. Crystallographic studies of SP-D lectin domains have shown that neither of these residues coordinates with calcium; however, their side chains can participate in hydrogen bonds with various bound ligands (14Crouch E. McDonald B. Smith K. Roberts M. Mealy T. Seaton B. Head J. Biochemistry. 2007; 46: 5160-5169Crossref PubMed Scopus (28) Google Scholar, 15Wang H. Head J. Kosma P. Brade H. Müller-Loennies S. Sheikh S. McDonald B. Smith K. Cafarella T. Seaton B. Crouch E. Biochemistry. 2008; 47: 710-720Crossref PubMed Scopus (54) Google Scholar, 16Shrive A.K. Tharia H.A. Strong P. Kishore U. Burns I. Rizkallah P.J. Reid K.B. Greenhough T.J. J. Mol. Biol. 2003; 331: 509-523Crossref PubMed Scopus (97) Google Scholar, 20Crouch E. McDonald B. Smith K. Cafarella T. Seaton B. Head J. J. Biol. Chem. 2006; 281: 18008-18014Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 21Crouch E. Hartshorn K. Horlacher T. McDonald B. Smith K. Cafarella T. Seaton B. Seeberger P.H. Head J. Biochemistry. 2009; 48: 3335-3345Crossref PubMed Scopus (55) Google Scholar, 22Håkansson K. Lim N.K. Hoppe H.J. Reid K.B. Structure Fold Des. 1999; 7: 255-264Abstract Full Text Full Text PDF Scopus (126) Google Scholar, 23Shrive A.K. Martin C. Burns I. Paterson J.M. Martin J.D. Townsend J.P. Waters P. Clark H.W. Kishore U. Reid K.B. Greenhough T.J. J. Mol. Biol. 2009; 394: 776-788Crossref PubMed Scopus (21) Google Scholar). Furthermore, differing interactions with these side chains contribute to known differences in saccharide selectivity. For example, the preference of human SP-D for d-N-acetyl-mannosamine over d-mannose can be attributed to hydrogen bonding of the Asp-325 carboxyl group with the N-acetyl substituent of the sugar (15Wang H. Head J. Kosma P. Brade H. Müller-Loennies S. Sheikh S. McDonald B. Smith K. Cafarella T. Seaton B. Crouch E. Biochemistry. 2008; 47: 710-720Crossref PubMed Scopus (54) Google Scholar, 24Crouch E.C. Smith K. McDonald B. Briner D. Linders B. McDonald J. Holmskov U. Head J. Hartshorn K. Am. J. Respir. Cell Mol. Biol. 2006; 35: 84-94Crossref PubMed Scopus (57) Google Scholar).TABLE 1Residues at positions 325 and 343 in SP-Ds and other collectinsPosition 325Position 343Surfactant protein D HumanAspArg D325A D325N R343V D325A/R343V Rat/mouseAsnLys CowAspLys PigAsnLysBovine serum collectins ConglutininSerVal CL-43ArgIle CL-46AsnValHuman MBP (MBP-C)AlaValSurfactant protein A HumanArgArg RatGlnArg Open table in a new tab MBP and the bovine serum collectins are characterized by amino acids with short side chains at the 325 position, and CL-43 shows a distinctive insertion that begins with arginine at this position (Table 1). We have previously demonstrated small increases in anti-viral activity with similar insertions of three amino acids (RAK or AAA) in the context of the wild-type human NCRD (25Crouch E. Tu Y. Briner D. McDonald B. Smith K. Holmskov U. Hartshorn K. J. Biol. Chem. 2005; 280: 17046-17056Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26Hartshorn K.L. White M.R. Smith K. Sorensen G. Kuroki Y. Holmskov U. Head J. Crouch E.C. Scand. J. Immunol. 2010; 72: 22-30PubMed Google Scholar). In addition, our previous studies have shown that residue 343 (arginine in humans or lysine in rodents and other species; Table 1) contributes to the binding of SP-D to phosphatidylinositol, the major surfactant-associated ligand (14Crouch E. McDonald B. Smith K. Roberts M. Mealy T. Seaton B. Head J. Biochemistry. 2007; 46: 5160-5169Crossref PubMed Scopus (28) Google Scholar), and to the inner core oligosaccharide domain of rough bacterial lipopolysaccharides (LPS) (15Wang H. Head J. Kosma P. Brade H. Müller-Loennies S. Sheikh S. McDonald B. Smith K. Cafarella T. Seaton B. Crouch E. Biochemistry. 2008; 47: 710-720Crossref PubMed Scopus (54) Google Scholar). In fact, the greater capacity of rodent SP-D to recognize these particular ligands is largely determined by the structure of the 343 side chain (14Crouch E. McDonald B. Smith K. Roberts M. Mealy T. Seaton B. Head J. Biochemistry. 2007; 46: 5160-5169Crossref PubMed Scopus (28) Google Scholar, 15Wang H. Head J. Kosma P. Brade H. Müller-Loennies S. Sheikh S. McDonald B. Smith K. Cafarella T. Seaton B. Crouch E. Biochemistry. 2008; 47: 710-720Crossref PubMed Scopus (54) Google Scholar). More recently, we have shown that the substitution of Arg-343 with valine (R343V), as found in MBP or conglutinin (Table 1), substantially increases interactions with certain mannose-rich oligosaccharides, enhances viral binding and neutralization, and confers the capacity to mediate viral aggregation (21Crouch E. Hartshorn K. Horlacher T. McDonald B. Smith K. Cafarella T. Seaton B. Seeberger P.H. Head J. Biochemistry. 2009; 48: 3335-3345Crossref PubMed Scopus (55) Google Scholar, 27Hartshorn K.L. White M.R. Tecle T. Sorensen G. Holmskov U. Crouch E.C. Am. J. Physiol. Lung Cell Mol. Physiol. 2010; 298: L79-L88Crossref PubMed Scopus (28) Google Scholar). Similar but less marked effects were observed when Arg-343 was substituted with isoleucine (R343I), as found in CL-43 (Table 1). In the present studies, we employed a novel surface plasmon resonance assay, viral binding and neutralization assays, mass spectrometry of HA glycans, and a new in vivo model of IAV infection to examine interactions between a recently developed combinatorial SP-D mutant and influenza A virus. The findings demonstrate the potential for collectin NCRD-based antiviral interventions. The pET-30a(+) vector, S-protein horseradish peroxidase, and RosettaBlue competent cells were from Novagen (Madison, WI). Fatty acid free BSA (BAH66-0050) was from Equitech-Bio, Inc, Kerrville, TX. All mono- and disaccharides were the d-anomers and were of the highest purity available from Sigma. Gel filtration protein standards were from Bio-Rad, and the HisCap Biosensors were from ICx Technologies, Oklahoma City, OK. The expression and characterization of the N-terminal-tagged, trimeric human neck+CRD (hNCRD) fusion proteins has been previously described (14Crouch E. McDonald B. Smith K. Roberts M. Mealy T. Seaton B. Head J. Biochemistry. 2007; 46: 5160-5169Crossref PubMed Scopus (28) Google Scholar, 20Crouch E. McDonald B. Smith K. Cafarella T. Seaton B. Head J. J. Biol. Chem. 2006; 281: 18008-18014Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 21Crouch E. Hartshorn K. Horlacher T. McDonald B. Smith K. Cafarella T. Seaton B. Seeberger P.H. Head J. Biochemistry. 2009; 48: 3335-3345Crossref PubMed Scopus (55) Google Scholar, 24Crouch E.C. Smith K. McDonald B. Briner D. Linders B. McDonald J. Holmskov U. Head J. Hartshorn K. Am. J. Respir. Cell Mol. Biol. 2006; 35: 84-94Crossref PubMed Scopus (57) Google Scholar, 25Crouch E. Tu Y. Briner D. McDonald B. Smith K. Holmskov U. Hartshorn K. J. Biol. Chem. 2005; 280: 17046-17056Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Site-directed mutagenesis was performed using a QuikChange II XL site-directed mutagenesis kit (200521; Stratagene, LA Jolla, CA). D325A was generated using the wild-type hSP-D neck+CRD DNA as template (25Crouch E. Tu Y. Briner D. McDonald B. Smith K. Holmskov U. Hartshorn K. J. Biol. Chem. 2005; 280: 17046-17056Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), whereas the D325A/R343V double mutant was generated using the cDNA for the previously characterized R343V mutant (14Crouch E. McDonald B. Smith K. Roberts M. Mealy T. Seaton B. Head J. Biochemistry. 2007; 46: 5160-5169Crossref PubMed Scopus (28) Google Scholar, 21Crouch E. Hartshorn K. Horlacher T. McDonald B. Smith K. Cafarella T. Seaton B. Seeberger P.H. Head J. Biochemistry. 2009; 48: 3335-3345Crossref PubMed Scopus (55) Google Scholar). Sequences were verified by automated sequencing of the entire coding sequence of the fusion protein. Glycerol stocks of transformed bacteria were stored in single-use aliquots at −80 °C. RosettaBlue competent cells were transformed with the wild-type or mutant construct in pET-30a(+) vector, and expressed proteins were isolated from inclusion bodies (25Crouch E. Tu Y. Briner D. McDonald B. Smith K. Holmskov U. Hartshorn K. J. Biol. Chem. 2005; 280: 17046-17056Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). After refolding and oligomerization, the D325A/R343V and D325A fusion proteins were purified by nickel-affinity chromatography, and trimers were isolated by gel filtration chromatography on an AKTA Purifier system. The purified proteins showed the expected mobility on SDS-PAGE (Fig. 1A, third and fourth lanes) with a slower migration in the presence of dithiothreitol, consistent with the formation of normal intrachain disulfide bonds, as illustrated for D325A/R343V (Fig. 1A, sixth lane). Protein concentrations were determined using the bicinchoninic (BCA) assay (Pierce) with BSA as a standard. Endotoxin levels were less than ∼3 pg/μg of protein, including preparations used for the in vivo experiments. Proteins were stored in small single-use aliquots at −80 °C. Analytical gel filtration confirmed the absence of aggregation of the purified trimers under our usual conditions of storage. Other NCRD constructs used for these experiments included R343V (21Crouch E. Hartshorn K. Horlacher T. McDonald B. Smith K. Cafarella T. Seaton B. Seeberger P.H. Head J. Biochemistry. 2009; 48: 3335-3345Crossref PubMed Scopus (55) Google Scholar), an expressed tagless wild-type human NCRD (hNCRD NoFT) (21Crouch E. Hartshorn K. Horlacher T. McDonald B. Smith K. Cafarella T. Seaton B. Seeberger P.H. Head J. Biochemistry. 2009; 48: 3335-3345Crossref PubMed Scopus (55) Google Scholar), and E321K, a human NCRD mutant that is defective in calcium coordination at the primary sugar binding site and shows no detectable lectin activity (28Carlson T.K. Torrelles J.B. Smith K. Horlacher T. Castelli R. Seeberger P.H. Crouch E.C. Schlesinger L.S. Glycobiology. 2009; 19: 1473-1484Crossref PubMed Scopus (18) Google Scholar). The Phil82 (H3N2) strain of IAV was grown and isolated as previously described (13Hartshorn K.L. Webby R. White M.R. Tecle T. Pan C. Boucher S. Moreland R.J. Crouch E.C. Scheule R.K. Respir. Res. 2008; 9: 65Crossref PubMed Scopus (84) Google Scholar). Binding of NCRD fusion proteins to solid-phase IAV, hemagglutination inhibition (HAI) assays, fluorescent focus assays of viral neutralization, and assays of viral aggregation and neutrophil uptake were performed as described in recent publications (13Hartshorn K.L. Webby R. White M.R. Tecle T. Pan C. Boucher S. Moreland R.J. Crouch E.C. Scheule R.K. Respir. Res. 2008; 9: 65Crossref PubMed Scopus (84) Google Scholar, 25Crouch E. Tu Y. Briner D. McDonald B. Smith K. Holmskov U. Hartshorn K. J. Biol. Chem. 2005; 280: 17046-17056Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In particular, binding of NCRD fusion protein to solid-phase viral particles was assessed using the S-protein horseradish peroxidase detection system; all assays were performed within the linear range of optical density. For some studies, findings for Phil82 were compared with SP-D resistant strains, such as Phil82BS and PR8 (H1N1) (13Hartshorn K.L. Webby R. White M.R. Tecle T. Pan C. Boucher S. Moreland R.J. Crouch E.C. Scheule R.K. Respir. Res. 2008; 9: 65Crossref PubMed Scopus (84) Google Scholar). The A/WSN/33 HAnc-Asp225Gly (H1N1) hybrid strain (hereafter designated WSNHAnc-Asp225Gly) was kindly provided by Dr. Donald Smee, Utah State University (29Smee D.F. Bailey K.W. Wong M.H. O'Keefe B.R. Gustafson K.R. Mishin V.P. Gubareva L.V. Antiviral Res. 2008; 80: 266-271Crossref PubMed Scopus (89) Google Scholar). We also examined the wild-type, mouse-adapted A/WSN/33 (H1N1) strain (hereafter designated WSN). HA molecules were released from Phil82 (H3N2), Phil82BS (H3N2), and PR8 (H1N1) virions using bromelain, a protease that cleaves the stalk of the trimeric hemagglutinin near the site of membrane insertion (30Wilson I.A. Skehel J.J. Wiley D.C. Nature. 1981; 289: 366-373Crossref PubMed Scopus (1993) Google Scholar). The purified bromelain-solubilized HAs were examined by SDS-PAGE in the absence and presence of dithiothreitol (Fig. 1B). The unreduced Phil82 protein migrated as a single major species of ∼70 kDa relative to globular standards; after reduction, there were two species of ∼61 and 25 kDa, consistent with the HA1 with HA2 subunits, respectively. Similar results were obtained for the Phil82BS HA (data not shown). As expected, the unreduced PR8 protein (61 kDa) and the PR8 HA1 subunit (53 kDa) migrated more rapidly than the corresponding Phil82 species, consistent with the absence of glycans on the HA1 subunit of the PR8 HA. The assembly of the purified HAs was assessed by gel filtration under non-denaturing conditions using an AKTA Tricorn 10/300 GL Superose 12 column (GE Healthcare) and a series of globular standards (Bio-Rad, #151-1901) (25Crouch E. Tu Y. Briner D. McDonald B. Smith K. Holmskov U. Hartshorn K. J. Biol. Chem. 2005; 280: 17046-17056Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The proteins eluted as single major peaks near the expected position of HA trimers, between thyroglobulin and γ-globulin standards (data not shown). The molecular mass was ∼250 kDa as estimated using a plot of elution volume versus log molecular weight plot, with the Phil82 or Phil82BS HA eluting very slightly earlier than the PR8 HA. Mass spectral data were acquired using an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA) interfaced with a nanoAcquity ultra performance liquid chromatograph (Waters Corp, Milford, MA) and a Triversa Nanomate robot (Advion Biosciences, Inc., Ithaca, NY). The protein (100 μg) was reduced using dithiothreitol and alkylated using iodoacetamide, desalted, and digested using trypsin using standard protocols. Using a reversed phase chromatography column (Waters BEH C18, 150 μm × 15 cm), liquid chromatography/tandem mass spectrometry data (LC/MS) were acquired using conditions as described (31Perlman D.H. Bauer S.M. Ashrafian H. Bryan N.S. Garcia-Saura M.F. Lim C.C. Fernandez B.O. Infusini G. McComb M.E. Costello C.E. Feelisch M. Circ. Res. 2009; 104: 796-804Crossref PubMed Scopus (46) Google Scholar), and the data were analyzed using the MASCOT Server software (Matrix Science, Inc. Boston, MA) operating in-house. The results were used to generate a custom data base of H3 type HA sequences for subsequent glycoproteomics analysis. An aliquot (10 μg) of the HA tryptic peptide mixture was digested with peptide N-glycosidase F (New England Biolabs, Andover, MA) in the presence of H218O and then analyzed by C18 reversed phase LC/MS as above with manual interpretations. For glycoproteomics, LC/MS was conducted using an in-house packed HILIC column (Amide-80, 3-μm beads, 250 μm × 15 cm, Tosoh Bioscience, Mongomeryville, PA) as described (32Naimy H. Buczek-Thomas J.A. Nugent M.A. Leymarie N. Zaia J. J. Biol. Chem. 2011; 286: 19311-19319Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Higher energy dissociation tandem MS scans containing oxonium ions corresponding to Hex and HexHexNAc triggered subsequent electron capture dissociation tandem mass spectra. Datasets were acquired using on-line LC/tandem MS. Additional data were acquired by collecting fractions from the HILIC column and analyzing off-line using the Triversa Nanomate robot. Candidate glycopeptides were identified based on the presence of 18O-Asp from manual interpretations. Glycopeptides were identified based on the presence of oxonium ions in higher energy dissociation scans and confirmed from the presence of neutral losses corresponding to Hex, HexNAc, NeuAc, and dHex residues in the higher energy dissociation scans. The peptide sequence was determined from the electron capture dissociation scans. Only those glycopeptides producing consistent proteomics and higher energy dissociation/electron capture dissociation analyses are reported (Table 2).TABLE 2Glycosylation of Phil82 HAGlycosylation siteHexHexNAcdHexTypeRelative amount14482M1881H/C0.3–0.4821H/C<0.0579H/C0.1–0.216572M<0.0182M0.10–0.2092M0.30–0.40102M<0.0162H/C<0.0563H/C<0.0573H/C0.40–0.5083H/C193H/C<0.05 Open table in a new tab SPR assays described in this publication were performed using a SensiQ Pioneer biosensing system and HisCap (Ni2+-nitrilotriacetic acid (NTA)) biosensors (ICx Technologies). However, certain preliminary experiments were performed using a BiaCore 2000 and the corresponding BiaCore Sensor Chip NTA (GE Healthcare). For the SensiQ, the usual configuration was to attach NCRD fusion proteins to cells 1 and/or 3, leaving cell 2 as a reference. Briefly, all cells were charged with 500 μm nickel chloride in 10 mm Hepes, 150 mm NaCl, pH 7.5 (HBS), resulting in a predictable change in refractive index. The trimeric hNCRDs were then coupled via their N-terminal hexahistidine tags to the desired flow cells, as recommended by the manufacturer, at a concentration of 1–10 μg/ml. For the experiments reported here protein couplings were performed in 10 mm sodium acetate, pH 5.0, to maximize attachment. Because all the proteins are stored at similar high concentrations in the same buffer, the coupling conditions were effectively identical. Preliminary experiments showed nearly equivalent changes in response units for equivalent amounts of several different trimeric NCRDs, indicating reproducible binding of the fusion proteins to the biosensor and confirming equivalence of the flow cells. Before the assay, the bound trimeric NCRD fusion proteins were “activated” by priming the system three times for a total 15 min with HBS containing 5 mm calcium chloride (HBS + C). The cells were then “blocked” with 0.1% fatty acid free bovine serum albumin in HBS + C, which helped minimize nonspecific binding to the reference cell. The initial rationale for calcifying the bound NCRDs was to preclude lectin-dependent binding to dextrans on the biosensor. Although this proved to be an unnecessary precaution, the approach facilitated experiments confirming the calcium-dependence of binding. Analytes such as bromelain-solubilized HAs were injected into the mobile phase at the indicated concentration (0.02–20 μg/ml) at a controlled temperature of 25 °C. Flow rates were optimized to minimize mass transport effects; 25 μl/min was used for the current studies. For some preliminary experiments, competing sugars were co-injected with the analyte to confirm specificity. However, for the experiments shown here a competing sugar was added during the dissociation and/or regeneration phase. The biosensors were routinely stripped with alternating injections of 350 mm EDTA in HBS, pH 8.0, and 1 m imidazole in deionized water. The stripped biosensors could be recharged with nickel and protein for multiple assays without deleterious effects on performance. Kinetic analyses and curve-fitting for the SensiQ were performed using Qdat (ICx Technologies). To facilitate formatting for figure preparation, data were exported to Sigmaplot (Systat Software). In vivo mortality studies were conducted with 8–12-week-old female DBA/2J mice (The Jackson Laboratory, Bar Harbor, ME). Mice were lightly anesthetized with isofluorane. 50 μl of WSN or WSNHAnc-Asp225Gly stock in DPBS with Ca2+ and Mg2+ ions was co-administered via intratracheal delivery with or without 5 μg/mouse D325A/R343V NCRD or wild-type NCRD. Viral titers of the inoculate were 1 × 105 and 3.3 × 105 viral particles/ml, respectively, as assessed by real-time quantitation (APM3600, EMD Millipore, Billerica, MA). Mice were weighed every other day and checked daily for viral induced mortality. All animals were maintained in a specific pathogen-free facility and were handled according to an institutional animal care and use committee (IACUC)-approved protocol and National Institutes of Health guidelines. In some experiments whole lungs were harvested 5 days post-viral infection (n = 3/group). The tissue was homogenized in DPBS without Ca2+ and Mg2+ and spun at 3000 × g for 10 min. The supernatant was collected and stored at −80 °C. Total RNA was isolated from infected mouse lung homogenate using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), and viral RNA was purified by the Viral RNA Spin Protocol (Qiagen) and stored at −20 °C. Real-time fluoresc" @default.
• W1970604893 created "2016-06-24" @default.
• W1970604893 creator A5007669776 @default.
• W1970604893 creator A5013242108 @default.
• W1970604893 creator A5018197827 @default.
• W1970604893 creator A5022829687 @default.
• W1970604893 creator A5029730540 @default.
• W1970604893 creator A5036151111 @default.
• W1970604893 creator A5051318955 @default.
• W1970604893 creator A5059963898 @default.
• W1970604893 creator A5085018824 @default.
• W1970604893 creator A5086486920 @default.
• W1970604893 creator A5088462075 @default.
• W1970604893 creator A5089080348 @default.
• W1970604893 creator A5089991342 @default.
• W1970604893 date "2011-11-01" @default.
• W1970604893 modified "2023-09-26" @default.
• W1970604893 title "Mutagenesis of Surfactant Protein D Informed by Evolution and X-ray Crystallography Enhances Defenses against Influenza A Virus in Vivo" @default.
• W1970604893 cites W142444126 @default.
• W1970604893 cites W1521344931 @default.
• W1970604893 cites W1553440365 @default.
• W1970604893 cites W1651702251 @default.
• W1970604893 cites W1976661013 @default.
• W1970604893 cites W1980528739 @default.
• W1970604893 cites W1984412128 @default.
• W1970604893 cites W1987273335 @default.
• W1970604893 cites W1990864962 @default.
• W1970604893 cites W1991051424 @default.
• W1970604893 cites W1999311908 @default.
• W1970604893 cites W2008308371 @default.
• W1970604893 cites W2019946911 @default.
• W1970604893 cites W2020317305 @default.
• W1970604893 cites W2028948043 @default.
• W1970604893 cites W2034073109 @default.
• W1970604893 cites W2036039932 @default.
• W1970604893 cites W2036411864 @default.
• W1970604893 cites W2038801195 @default.
• W1970604893 cites W2050299848 @default.
• W1970604893 cites W2060278327 @default.
• W1970604893 cites W2060441535 @default.
• W1970604893 cites W2063714476 @default.
• W1970604893 cites W2075488647 @default.
• W1970604893 cites W2076424086 @default.
• W1970604893 cites W2079118396 @default.
• W1970604893 cites W2080077176 @default.
• W1970604893 cites W2081297120 @default.
• W1970604893 cites W2081635099 @default.
• W1970604893 cites W2087993981 @default.
• W1970604893 cites W2098901782 @default.
• W1970604893 cites W2104063702 @default.
• W1970604893 cites W2111668132 @default.
• W1970604893 cites W2114710822 @default.
• W1970604893 cites W2114934116 @default.
• W1970604893 cites W2125782486 @default.
• W1970604893 cites W2148059071 @default.
• W1970604893 cites W2155498715 @default.
• W1970604893 cites W2162540544 @default.
• W1970604893 cites W2172063653 @default.
• W1970604893 cites W2399665621 @default.
• W1970604893 cites W2411620058 @default.
• W1970604893 doi "https://doi.org/10.1074/jbc.m111.300673" @default.
• W1970604893 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3220461" @default.
• W1970604893 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21965658" @default.
• W1970604893 hasPublicationYear "2011" @default.
• W1970604893 type Work @default.
• W1970604893 sameAs 1970604893 @default.
• W1970604893 citedByCount "36" @default.
• W1970604893 countsByYear W19706048932012 @default.
• W1970604893 countsByYear W19706048932013 @default.
• W1970604893 countsByYear W19706048932014 @default.
• W1970604893 countsByYear W19706048932015 @default.
• W1970604893 countsByYear W19706048932016 @default.
• W1970604893 countsByYear W19706048932017 @default.
• W1970604893 countsByYear W19706048932018 @default.
• W1970604893 countsByYear W19706048932019 @default.
• W1970604893 countsByYear W19706048932020 @default.
• W1970604893 countsByYear W19706048932021 @default.
• W1970604893 countsByYear W19706048932022 @default.
• W1970604893 countsByYear W19706048932023 @default.
• W1970604893 crossrefType "journal-article" @default.
• W1970604893 hasAuthorship W1970604893A5007669776 @default.
• W1970604893 hasAuthorship W1970604893A5013242108 @default.
• W1970604893 hasAuthorship W1970604893A5018197827 @default.
• W1970604893 hasAuthorship W1970604893A5022829687 @default.
• W1970604893 hasAuthorship W1970604893A5029730540 @default.
• W1970604893 hasAuthorship W1970604893A5036151111 @default.
• W1970604893 hasAuthorship W1970604893A5051318955 @default.
• W1970604893 hasAuthorship W1970604893A5059963898 @default.
• W1970604893 hasAuthorship W1970604893A5085018824 @default.
• W1970604893 hasAuthorship W1970604893A5086486920 @default.
• W1970604893 hasAuthorship W1970604893A5088462075 @default.
• W1970604893 hasAuthorship W1970604893A5089080348 @default.
• W1970604893 hasAuthorship W1970604893A5089991342 @default.
• W1970604893 hasBestOaLocation W19706048931 @default.
• W1970604893 hasConcept C104317684 @default.
• W1970604893 hasConcept C121332964 @default.
• W1970604893 hasConcept C136449434 @default.
• W1970604893 hasConcept C159047783 @default.

• next