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Large scale identification of proteins, mucins, and their O-glycosylation in the endocervical mucus during the menstrual cycle.

Journal article
Authors Ylva Andersch-Björkman
Kristina A Thomsson
Jessica Holmén Larsson
Erling Ekerhovd
Gunnar C. Hansson
Published in Molecular & cellular proteomics : MCP
Volume 6
Issue 4
Pages 708-16
ISSN 1535-9476
Publication year 2007
Published at Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Institute of Clinical Sciences
Pages 708-16
Language en
Links dx.doi.org/10.1074/mcp.M600439-MCP2...
Keywords Adult, Carbohydrate Sequence, Cervix Mucus, chemistry, Female, Glycosylation, Humans, Menstrual Cycle, metabolism, Molecular Sequence Data, Mucins, chemistry, isolation & purification, Proteins, chemistry, isolation & purification, Proteomics, Tandem Mass Spectrometry
Subject categories Medical and Health Sciences

Abstract

The mucus filling the human cervical opening blocks the entry to the uterus, but this has to be relative and allow for the sperm to penetrate at ovulation. We studied this mucus, its content of proteins and mucins, and the mucin O-glycosylation in cervical secretions before, during, and after ovulation. Cervical mucosal secretions from 12 subjects were collected, reduced-alkylated, separated with polyacrylamide or agarose/polyacrylamide gel electrophoresis, and stained with silver, Alcian blue, or Coomassie Blue stain. Protein and mucin bands from before and during ovulation were digested and subsequently analyzed by nano-LC-FT-ICR MS and MS/MS. We identified 194 proteins after searches against the NCBI non-redundant protein database and an in-house mucin database. Three gel-forming (MUC5B, MUC5AC, and MUC6) and two transmembrane mucins (MUC16 and MUC1) were identified. For the analysis of mucin O-glycosylation, separated mucins from six individuals were blotted to PVDF membranes, and the O-glycans were released by reductive beta-elimination and analyzed with capillary HPLC-MS and -MS/MS. At least 50 neutral, sialic acid-, and sulfate-containing oligosaccharides were found. An increase of GlcNAc-6GalNAcol Core 2 structures and a relative decrease of NeuAc residues are typical for ovulation, and NeuAc-6GalNAcol and NeuAc-3Gal- epitopes are typical for the non-ovulatory phases. The cervical mucus at ovulation is thus characterized by a relative increase in neutral fucosylated oligosaccharides. This comprehensive characterization of the mucus during the menstrual cycle suggests mucin glycosylation as the major alteration at ovulation, but the relation to the altered physicochemical properties and sperm penetrability is still not understood.

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