The Carbohydrate Structure of DEFB126, the Major Component of the Cynomolgus Macaque Sperm Plasma Membrane Glycocalyx |
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Authors: | A?I?Yudin C?A?Treece T?L?Tollner J?W?Overstreet Email author" target="_blank">G?N?CherrEmail author |
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Institution: | (1) Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of California, Davis, CA, USA;(2) Center for the Health and Environment, University of California, Davis, CA, USA;(3) Departments of Environmental Toxicology and Nutrition, University of California, Davis, CA, USA;(4) Bodega Marine Laboratory, University of California, Davis, CA, USA |
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Abstract: | Based on the amino-acid sequence of the macaque epididymal secretory protein, ESP 13.2 (Q9BEE3/AJ236909), it has now been
classified as β-defensin DEFB126. DEFB126 is one of the five β-defensins with genes that are clustered along chromosome 20pl3,
and all five proteins have an extended carboxy terminus that continues beyond the 6-cysteine β-defensin core region. This
60-amino acid carboxyl tail extension of the DEFB126 molecule is extraordinarily rich in threonine and serine (40%), many
of which appear to be likely candidates for having O-glycosylation. DEFB126 has been shown to coat the entire surface of cynomolgus
macaque sperm as they move through the corpus/caudal region of the epididymis. It is a major glycocalyx barrier to the external
environment and is retained until the completion of capacitation. Sperm exposed to fluorescein-conjugated poly-L-lysine or Alexa488-histone showed a very uniform fluorescent labeling pattern over the entire sperm surface, almost identical
to that observed with anti-DEFB126 Ig label. Sperm surface components that were released following treatment with caffeine/cAMP
(in vitro capacitation) were blotted and probed with three different lectins which are known to recognize terminal sialic
acid residues, and all three recognized the 35 kDa DEFB126 band. Neuraminidase treatment of sperm shifted the molecular weight
of DEFB126 from 34–36 kDa to approximately 38–40 kDa and removed or greatly inhibited sialic acid-specific lectin recognition.
O-Glycanase treatment alone was ineffective at removal of the oligosaccharides, but prior treatment with neuraminidase was
sufficient to enable the O-glycanase treatment to effectively change the apparent molecular weight to 10 kDa, confirming that
a major portion of the molecular mass is associated with the carbohydrate portion. Western blots of neuraminidase-treated
DEFB126 showed strong recognition with a number of lectins that identify β-galactose and also lectins that recognize the N-acetylgalactosamine-serine/threonine,
the proposed connection site of O-glycosylation. All of the lectins that recognized DEFB126 on Western blots were used to
fluorescently probe sperm. The fluorescent patterns that were observed with poly-L-lysine, Alexa488-histone, sialic acid-specific lectins, and galactose-specific lectins showed even distributions over the
entire sperm surface and the patterns were identical to sperm labeled with anti-DEFB126 Ig, and all but the antibody did not
recognize neuraminidase-treated sperm. |
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Keywords: | Sperm β -Defensin Glycocalyx Sialic acid DEFB126 |
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