Altered Carbohydrate Recognition Specificity Engineered into Surfactant Protein D Reveals Different Binding Mechanisms for Phosphatidylinositol and Glucosylceramide

ABSTRACT Collectins play important roles in host defense against infectious microorganisms. We now demonstrate that the serum collectins mannose-binding lectin (MBL) and conglutinin have less ability to bind to, aggregate, and enhance neutrophil uptake of several strains of gram-negative and gram-positive bacteria than pulmonary surfactant protein D (SP-D). Collectins are composed of four major structural domains (i.e., N-terminal, collagen, and neck and carbohydrate recognition domains). To determine which domains of SP-D are responsible for its greater bacterial binding or aggregating activity, activities of chimeric collectins containing the N-terminal and collagen domains of SP-D coupled to the neck recognition domains and carbohydrate recognition domains (CRD) of MBL or conglutinin (SP-D/Congneck+CRD and SP-D/MBLneck+CRD) were tested. The SP-D/Congneck+CRD and SP-D/MBLneck+CRD chimeras bound to and aggregated the bacteria more strongly than did wild-type MBL or conglutinin. SP-D/MBLneck+CRD also enhanced neutrophil uptake of bacteria more so than MBL. Hence, the SP-D N-terminal and/or collagen domains contribute to the enhanced bacterial binding and aggregating activities of SP-D. In prior studies, SP-D/Congneck+CRD and SP-D/MBLneck+CRD had increased ability to bind to influenza virus compared not only with that of conglutinin or MBL but with that of wild-type SP-D as well. In contrast, the chimeras had either reduced or unchanged ability to bind to or aggregate bacteria compared to that of wild-type SP-D. Hence, although replacement of the neck recognition domains and CRDs of SP-D with those of MBL and conglutinin conferred increased viral binding activity, it did not favorably affect bacterial binding activity, suggesting that requirements for optimal collectin binding to influenza virus and bacteria differ.

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Expression of the Carbohydrate Recognition Domain of Lung Surfactant Protein D and Demonstration of Its Binding to Lipopolysaccharides of Gram-Negative Bacteria

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1994.2127 ◽

1994 ◽

Vol 202 (3) ◽

pp. 1674-1680 ◽

Cited By ~ 85

Author(s):

B.L. Lim ◽

J.Y. Wang ◽

U. Holmskov ◽

H.J. Hoppe ◽

K.B.M. Reid

Keyword(s):

Lung Surfactant ◽

Surfactant Protein ◽

Surfactant Protein D ◽

Carbohydrate Recognition Domain ◽

Gram Negative Bacteria ◽

Carbohydrate Recognition ◽

Gram Negative ◽

Lung Surfactant Protein

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Identification of calcium-dependent epitopes in the region of the carbohydrate recognition domain of lung surfactant protein D responsible for the interaction with gp-340

Immunology Letters ◽

10.1016/s0165-2478(97)87293-8 ◽

1997 ◽

Vol 56 (1-3) ◽

pp. 115

Author(s):

U Holmskov

Keyword(s):

Lung Surfactant ◽

Surfactant Protein ◽

Surfactant Protein D ◽

Carbohydrate Recognition Domain ◽

Carbohydrate Recognition ◽

Calcium Dependent ◽

Lung Surfactant Protein

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Monoclonal antibody-assisted structure-function analysis of the carbohydrate recognition domain of surfactant protein D

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00096.2010 ◽

2010 ◽

Vol 299 (3) ◽

pp. L384-L392 ◽

Cited By ~ 7

Author(s):

Kevan L. Hartshorn ◽

Mitchell R. White ◽

Michael Rynkiewicz ◽

Grith Sorensen ◽

Uffe Holmskov ◽

...

Keyword(s):

Antiviral Activity ◽

Binding Site ◽

Influenza A ◽

Function Analysis ◽

Specific Binding ◽

Surfactant Protein ◽

Receptor Protein ◽

Surfactant Protein D ◽

Carbohydrate Recognition Domain ◽

Carbohydrate Recognition

Surfactant protein D (SP-D) plays important roles in host defense against a variety of pathogens including influenza A virus (IAV). Ligand binding by SP-D is mediated by the trimeric neck and carbohydrate recognition domain (NCRD). We used monoclonal antibodies (mAbs) against human SP-D and a panel of mutant collectin NCRD constructs to identify functionally and structurally important epitopes. The ability of SP-D to bind to IAV and mannan involved partially overlapping binding sites that are distinct from those involved in binding to the glycoprotein-340 (gp-340) scavenger receptor protein. A species-specific motif (D324,D325,R343), which has been implicated in the specific binding of several ligands, contributes to recognition by mAbs that block antiviral or mannan binding activity. D325, in particular, is involved in the epitopes of these blocking mAbs. Conversely, the interspecies substitution of arginine for Lys343 in the rat NCRD (rK343R) conferred binding to two of the mAbs. The single site substitution of alanine for R349 or E347 resulted in highly selective alterations in mAb binding and caused decreased antiviral activity. Mutations at Glu333 (E333A), Trp340 (W340F), and Phe335 (F335A), which abrogated antiviral activity, were associated with decreased binding to multiple blocking mAbs, consistent with critical structural roles. More conservative substitutions at 335, which showed a significant increase in neutralization activity, caused selective loss of binding to one mAb. The analysis reveals, for the first time, an extended binding site for IAV; calcium-dependent antiviral activity involves residues flanking the primary carbohydrate binding site as well as more remote residues displayed on the carbohydrate recognition domain surface.

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