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Electron microscopy of cervical-mucus glycoproteins and fragments therefrom. The use of colloidal gold to make visible 'naked' protein regions.

ABSTRACT: Subunits of human cervical-mucus glycoproteins obtained by reductive cleavage of whole mucins and high-Mr glycopeptides (T-domains) obtained after their trypsin digestion were studied with electron microscopy after spreading the macromolecules in a monolayer of benzyldimethylalkylammonium chloride. Subunits were observed as linear and apparently flexible particles, with number- and weight-average lengths of 390 nm and 460 nm respectively. T-domains randomly distributed on the grid have number- and weight-average lengths of 90 nm and 103 nm respectively, whereas when aligned (possibly stretched by flow) they were longer, with number-average and weight-average lengths of 150 nm and 170 nm respectively. Subunits complexed with gold appeared as segmented structures, with a distribution of inter-gold distances similar to the length distribution for the relaxed T-domains. The whole mucins had few binding sites for gold, suggesting that reduction exposes hydrophobic protein-rich regions with high affinity for gold. Most T-domains had a binding site at one end, indicating the presence of a residual protruding naked peptide region. We conclude that mucins are assembled from subunits joined end-to-end, and that each subunit consists of alternating oligosaccharide 'clusters' (approx. 100 nm) and naked peptide regions which have (after reduction) a high affinity for colloidal gold.

SUBMITTER: Sheehan JK

PROVIDER: S-EPMC1136627 | biostudies-other | 1990 Jan

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Endometriosis is a chronic gynecological inflammatory disease characterized by the presence of functional endometrial glands and stroma outside of the uterine cavity. It affects 7-10% of women of reproductive age and up to 50% of women with infertility. The current gold standard for the diagnosis combines laparoscopic evaluation and biopsy of the visualized lesions. However, laparoscopy requires general anesthesia and developed surgical skills and it has a high procedural cost. In addition, it is associated with the risk, although rare, of potential intraoperative or postoperative complications. To date, several noninvasive biomarkers have been proposed; however, no definite diagnostic biomarker is yet available. The aim of this study was to characterize the CM proteome in patients with endometriosis using high resolution mass spectrometry-based proteomics, implemented by bioinformatic tools for quantitative analysis, in order to investigate the pathophysiological mechanisms of endometriosis. METHODS:Cervical mucus samples were collected from patients affected by endometriosis and fertile controls. An aliquot of the soluble acidic fraction of each cervical mucus sample, corresponding to 0.5 mg of total protein, was left to digest with sequencing grade modified porcine trypsin. The peptides were analyzed by LC-MS/MS on a high resolution Orbitrap Elite mass spectrometer and data were evaluated using bioinformatic tools. RESULTS:We aimed at the first total profiling of the cervical mucus proteome in endometriosis. From the list of identified proteins, we detected a number of differentially expressed proteins, including some functionally significant proteins. Six proteins were quantitatively increased in endometriosis, almost all being involved in the inflammatory pattern. Nine proteins were quantitatively reduced in endometriosis, including some proteins related with local innate immunity (CRISP-3 and Pglyrp1) and protection against oxidative stress (HSPB1). Fifteen proteins were not detected in endometriosis samples including certain proteins involved in antimicrobial activity (SLURP1 and KLK13) and related to seminal plasma liquefaction and male fertility (KLK13). CONCLUSIONS:This is the first application of high resolution mass spectrometry-based proteomics aimed in detecting an array of proteins in CM to be proposed for the noninvasive diagnosis of endometriosis. This chronic disease presents in CM an inflammatory protein pattern.

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Electron microscopy of cervical-mucus glycoproteins and fragments therefrom. The use of colloidal gold to make visible 'naked' protein regions.

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