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Streptococcus agalactiae CAMP factor binds to GPI-anchored proteins

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Abstract

CAMP factor (protein B) is a pore-forming protein secreted by Streptococcus agalactiae. It causes lysis of sheep red blood cells when these have been sensitized with staphylococcal sphingomyelinase. We here show that CAMP factor binds to GPI-anchored proteins, and that this interaction involves the carbohydrate core of the GPI-anchor. Enzymatic cleavage of GPI-anchors with phosphatidylinositol-specific phospholipase C strongly reduces the sensitivity of erythrocytes to CAMP factor. Incorporation of alkaline phosphatase, a model GPI-anchored protein, into liposome membranes renders the latter susceptible to permeabilization by CAMP factor. GPI-anchored proteins therefore function as cellular receptors for CAMP factor.

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Abbreviations

BAC-SulfoNHS:

Biotinamidohexanoic acid 3-sulfo-N-hydroxysulfosuccinimide ester

C16-GPI:

6-O-{{6-O-[N-(l-prolyl-l-seryl)-2-aminoethyl]-phosphonato-α-d-mannopyranosyl}-(1,2)-O-(α-d-mannopyranosyl)-(1,6)-O-(α-d-mannopyranosyl)-(1,4)-O-(2-amino-2-deoxy-α-d-glucopyranosyl)}-2-O-hexadecanoyl-myo-inositol

CHAPS:

3-[(3-Cholamido propyl)-dimethylammonio]-1-propanesu1fonate

DMSO:

Dimethylsulfoxide

DPPC:

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine

GPI:

Glycosylphosphatidylinositol

HBS:

20 mM HEPES, 150 mM NaCl, 10 mM MgCl2, pH 7.5

PBS:

20 mM potassium phosphate, 150 mM NaCl, pH 7.5

PI-PLC:

Phosphatidylinositol-specific phospholipase C

PLAP:

Placental alkaline phosphatase

PMSF:

Phenylmethylsulfonylfluoride

SDS-PAGE:

Sodium dodecylsulfate polyacrylamide gel electrophoresis

TBS:

100 mM Tris, 0.9% NaCl, pH 7.5

TBS-Tween:

TBS with 0.1% (v/v) Tween 20

TMB:

3,3′,5,5′-Tetramethylbenzidine

Triton-HEPES:

20 mM HEPES (pH 7.5) containing 0.06% Triton X-100

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Acknowledgments

This work was supported by NSERC (grant No. 250265–2003 GSC-032). The synthesis of C16-GPI was supported by an NSF grant (CHE-0554777).

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Authors and Affiliations

  1. Department of Chemistry, University of Waterloo, N2L 3G1, Waterloo, ON, Canada

    Shenhui Lang & Michael Palmer

  2. Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI, 48202, USA

    Jie Xue & Zhongwu Guo

Authors
  1. Shenhui Lang
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  2. Jie Xue
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  3. Zhongwu Guo
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  4. Michael Palmer
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Correspondence to Michael Palmer.

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Lang, S., Xue, J., Guo, Z. et al. Streptococcus agalactiae CAMP factor binds to GPI-anchored proteins. Med Microbiol Immunol 196, 1–10 (2007). https://doi.org/10.1007/s00430-006-0021-2

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