Human spermatozoa contain multiple targets for protein S-nitrosylation: an alternative mechanism of the modulation of sperm function by nitric oxide?
Nitric oxide (NO) enhances human sperm motility and capacitation associated with increased protein phosphorylation. NO activates soluble guanylyl cyclase, but can also modify protein function covalently via S-nitrosylation of cysteine. Remarkably, this mechanism remains unexplored in sperm although they depend on post-translational protein modification to achieve changes in function required for fertilisation. Our objective was to identify targets for S-nitrosylation in human sperm. Spermatozoa were incubated with NO donors and S-nitrosylated proteins were identified using the biotin switch assay and a proteomic approach using MS/MS. 240 S-nitrosylated proteins were detected in sperm incubated with S-nitroso-glutathione. Minimal levels were observed in glutathione or untreated samples. Proteins identified consistently based on multiple peptides included established targets for S-nitrosylation in other cells e.g. tubulin, GST and HSPs but also novel targets including A-kinase anchoring protein (AKAP) types 3 and 4, voltage-dependent anion-selective channel protein 3 and semenogelin 1 and 2. In situ localisation revealed S-nitrosylated targets on the postacrosomal region of the head and throughout the flagellum. Potential targets for S-nitrosylation in human sperm include physiologically significant proteins not previously reported in other cells. Their identification will provide novel insight into the mechanism of action of NO in spermatozoa.
Reproductive Biology and Genetics Group, Division of Reproductive and Child Health, The Medical School, University of Birmingham, Edgbaston, Birmingham, UK.
Linda
L
Lefièvre
Yongjian
Y
Chen
Sarah J
SJ
Conner
Joanna L
JL
Scott
Steve J
SJ
Publicover
W Christopher L
WC
Ford
Christopher L R
CL
Barratt
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D008297:Male; D033681:Mitochondrial Membrane Transport Proteins; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000378:metabolism / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D009603:Nitroso Compounds / Q000378:metabolism*; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D026422:S-Nitrosoglutathione / Q000378:metabolism / Q000494:pharmacology; D029607:Seminal Vesicle Secretory Proteins / Q000378:metabolism; D013094:Spermatozoa / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D050994:Voltage-Dependent Anion Channels / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D033681:Mitochondrial Membrane Transport Proteins; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D020543:Proteome; D029607:Seminal Vesicle Secretory Proteins; C494768:VDAC3 protein, human; D050994:Voltage-Dependent Anion Channels; C424482:seminal vesicle-specific antigen; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D052250:Nitric Oxide Synthase Type III; D003545:Cysteine