Steroids regulate α2,6-sialic acid-containing glycoconjugates in murine uterine epithelium at the time of implantation

Sialic acids are involved in many cellular interactions. They can serve as an adhesion ligand or act as an inhibitor to cellular adhesion by charge repulsion or by masking potential ligands. Although sialic acids are implicated in the process of blastocyst implantation, their expression and regulation in uterine epithelium of mice have not been studied. The lectin, Sambucus nigra (SNA) specifically recognizes α2,6-linked sialic acids, which are involved in cell recognition phenomena. It was used to probe frozen uterine sections from mice during days one through six of pregnancy. SNA staining was most intense at the apical surface of uterine epithelial cells on day one of pregnancy, decreased gradually through day four, and was undetectable by day five. The role of the steroid hormones, estrogen and progesterone, in regulating the expression of α2,6-linked sialic acids was studied in uterine sections from mice during the estrous cycle and in ovariectomized mice given hormone replacement using SNA. SNA staining of these sections during the estrous cycle showed that the expression of α2,6-linked sialic acids was stage dependent. Staining was most intense in uterine sections from mice in estrus, and was not detected in sections from mice in diestrus. In ovariectomized mice, staining was most intense in mice injected with estradiol alone, and no staining was evident in mice injected with progesterone alone. These results suggest that the expression of α2,6-linked sialic acids decreases during the time of implantation and that estrogen stimulates and progesterone inhibits its expression. β-Galactoside α2,6-Sialyltransferase (α2,6-ST) is the enzyme that links sialic acids to Galβ1-4GlcNAc termini of N-linked oligosaccharides. In order to investigate the mechanism behind the hormonal regulation of α2,6-linked sialic acids, the expression of α2,6-ST was followed in uterine sections from mice during early pregnancy, during the estrous cycle, and in ovariectomized mice given hormone replacement. In-situ hybridization was performed using digoxigenin labeled RNA probes to characterize α2,6-ST mRNA levels in uterine sections. Expression of α2,6-ST protein was also measured in uterine sections with a polyclonal antibody against α2,6-ST. The expression of α2,6-ST mRNA and protein correlated well with the timing of the appearance of α2,6-linked sialic acids. These results show that the expression of α2,6-linked sialic acids on the surface of mouse uterine epithelium decreases at the time of implantation and furthermore, that this decrease is due to the regulation of α2,6-ST by the steroid hormones. α2,6-linked sialic acids may serve to inhibit cellular adhesion by creating a charge repulsion, or by masking potential binding sites. Removal of this inhibition may permit blastocyst implantation

Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry

Oxidation of methionine residues in biopharmaceuticals is a common and often unwanted modification that frequently occurs during their manufacture and storage. It often results in a lack of stability and biological function of the product, necessitating continuous testing for the modification throughout the product shelf life. A major class of biopharmaceutical products are monoclonal antibodies (mAbs), however, techniques for their detailed structural analysis have until recently been limited. Hydrogen/deuterium exchange mass spectrometry (HXMS) has recently been successfully applied to the analysis of mAbs. Here we used HXMS to identify and localise the structural changes that occurred in a mAb (IgG1) after accelerated oxidative stress. Structural alterations in a number of segments of the Fc region were observed and these related to oxidation of methionine residues. These included a large change in the hydrogen exchange profile of residues 247–253 of the heavy chain, while smaller changes in hydrogen exchange profile were identified for peptides that contained residues in the interface of the CH2 and CH3 domains