Quantitative analysis and process monitoring of site-specific glycosylation microheterogeneity in recombinant human interferon-γ from Chinese hamster ovary cell culture by hydrophilic interaction chromatography

A chromatographic method was developed for quantitative analysis of site-specific microheterogeneity of the two N-linked glycosylation sites in recombinant human interferon-γ produced from Chinese hamster ovary (CHO) cell culture. After the interferon-γ was harvested by affinity chromatography, the tryptic digestion was carried out. The two glycopeptide pools, isolated from reversed-phase chromatography of tryptic digestion of interferon-γ, were subjected to further separation by hydrophilic interaction chromatography. Each peak in the chromatograms was identified by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI–TOF–MS). The overall elution order of the glycopeptides was the following: neutral glycopeptides, monosialylated glycopeptides, bisialylated glycopeptides, trisialylated glycopeptide and tetrasialylated glycopeptides. Based on the integrated peak area for each compound in the chromatograms, the percentage for each glycan was utilized to quantify the glycosylation pattern of the interferon-γ. Finally, sialylation and antennarity structure percentages at the two glycosylation sites were chosen as the quality indicators in process monitoring of interferon-γ production from a serum-free suspension-batch CHO culture.     

Catalytic properties of DNA polymerase α activity associated with the heat-stabilized nuclear matrix prepared from HeLa S3 cells

We have investigated whether or not ATP or other nucleoside di- and trisphosphates (including some nonhydrolysable ATP analogues) can stimulate the activity and/or the processivity of DNA polymerase α associated with the nuclear matrix obtained from HeLa S3 cell nuclei that had been stabilized at 37°C prior to subfractionation, as has been reported previously for DNA polymerase α bound to the nuclear matrix prepared from 22-h regenerating rat liver. We have found that HeLa cell matrix-associated DNA polymerase α activity could not be stimulated at all by ATP or other nucleotides, a behaviour which was shared also by DNA polymerase α activity that solubilizes from cells during the isolation of nuclei and that is thought to be a form of the enzyme not actively engaged in DNA replication. Moreover, the processivity of matrix-bound DNA polymerase α activity was low (< 10 nucleotides). These results were obtained with the matrix prepared with either 2M NaCl or 0·25 M (NH₄)₂SO₄ and led us to consider that a 37° incubation of isolated nuclei renders resistant to high-salt extraction a form of DNA polymerase α which is unlikely to be involved in DNA replication in vivo.     

Adhesion and protease activity in cell lines from human salivary gland tumors are regulated by the laminin-derived peptide AG73, syndecan-1 and β1 integrin

We studied the induction of protease activity by the laminin alpha1-derived peptide AG73 in cells from adenoid cystic carcinoma (CAC2) and myoepithelioma (M1), respectively a malignant and a benign salivary gland tumors. Laminin alpha1 chain and MMP9 were immunolocalized in adenoid cystic carcinoma and myoepithelioma in vivo and in vitro. Cells grown inside AG73-enriched laminin-111 exhibited large spaces in the extracellular matrix, suggestive of remodeling. The broad spectrum MMP inhibitor GM6001 decreased spaces induced by AG73 in CAC2 and M1 cells. This result strongly suggests that AG73-mediated matrix remodeling involves matrix metalloproteinases. CAC2 and M1 cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, as detected by zymography. Furthermore, siRNA silencing of MMP9 decreased remodeling in 3D cultures. We searched for AG73 receptors regulating MMP9 activity in our cell lines. CAC2 and M1 cells grown on AG73 exhibited colocalization of syndecan-1 and beta1 integrin. siRNA knockdown of syndecan-1 expression in these cells resulted in decreased adhesion to AG73 and reduced protease and remodeling activity. We investigated syndecan-1 co-receptors in both cell lines. Silencing beta1 integrin inhibited adhesion to AG73, matrix remodeling and protease activity. Double-knockdown experiments were carried out to further explore syndecan-1 and beta1 integrin cooperation. CAC2 cells transfected with both syndecan-1 and beta1 integrin siRNA oligos showed significant decrease in adhesion to AG73. Simultaneous silencing of receptors also induced a decrease in protease activity. Our results suggest that syndecan-1 and beta1 integrin signaling downstream of AG73 regulate adhesion and MMP production by CAC2 and M1 cells.