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The mechanism of thermal degradation of a high-molecular-weight glycoprotein complex from bovine cervical mucus.

ABSTRACT: Gel-like oestrus bovine cervical mucus can be brought to the point of dissolution by thermal treatment. The glycoprotein complex so produced was isolated on CsCl density gradients, and found to be of a size comparable with that of a complex purified from mucus that had been brought to the point of dissolution by mild mechanical stirring. The latter material (GP-S) had a mol.wt. of 15.9 X 10(6) and was used to study further the effect of thermal treatment. Time and temperature lead to a gradual breakdown of GP-S, which is characterized by a single activation energy of 93.3 kJ/mol (22.3 kcal/mol) over the temperature range of 21-99 degrees C. The process responsible is thermal hydrolysis of peptide bonds, particularly next to aspartic acid residues. This conclusion is consistent with the appearance of aspartic acid as a new N-terminal amino acid and the activation energy of the process. After thermal degradation there is an increase in the buoyant density of GP-S and a change in the amino acid composition. These findings were found to be consistent with the loss of the naked peptide region and the preponderance of aspartic acid residues in this region. Thermal degradation therefore does not involve dispersion of non-covalent bonds, and indeed GP-S is quite unaffected by media commonly used to disperse such bonds.

SUBMITTER: Meyer FA

PROVIDER: S-EPMC1154132 | biostudies-other | 1983 Mar

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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The mechanism of thermal degradation of a high-molecular-weight glycoprotein complex from bovine cervical mucus.

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