Biomedical subjects

Yoannis Imbert

Publications and source records attributed to Yoannis Imbert.

4 recordsLinked to original sources

PubMed2006

MUC1 splice variants in human ocular surface tissues: possible differences between dry eye patients and normal controls.

Mucins are highly glycosylated proteins that are vital to the maintenance of healthy epithelial surfaces including the ocular surface. Mucins act as lubricants, protectants, and mediators of signal transduction. The majority of the O-glycosylation sites on the transmembrane mucin MUC1 are found in a highly polymorphic core region containing a variable number of tandem repeats (VNTR). MUC1 alleles can be divided into size classes that contain small (30-45) or large (60-90) numbers of repeats. Although at least 12 splice variants of MUC1 have been found in other tissues, no splice variants have been reported in human ocular surface tissues. We have used RT-PCR to identify MUC1 splice variants that were then confirmed by sequencing. We here report the presence in some samples of human cornea, conjunctiva, and lacrimal gland of MUC1/B which features canonical splicing between exons 1 and 2 and MUC1/A, a transcript that retains 27bp from the 3' end of intron 1 and is predicted to add 9 amino acids to the MUC1 sequence upstream of the tandem repeat region. Cornea and conjunctiva both contain the MUC1/SEC splice variant that lacks the transmembrane domain and, therefore, results in a soluble, secreted form of MUC1. Cornea and conjunctiva also contain MUC1/Y and MUC1/Z(X) variants that lack the tandem repeat region. Cornea, conjunctiva, and lacrimal gland also contain a previously undescribed MUC1 variant transcript, termed MUC1/YI, that retains 99bp from the 5' end and 27bp from the 3' end of the first intron, resulting in a frame shift and premature stop codon. This transcript is predicted to produce a novel 27 amino acid peptide after signal peptidase cleavage. Analysis of brush cytology samples revealed that the percentage of dry eye patients expressing the MUC1/A variant in the conjunctival epithelium is lower than in normal control donors. Western blotting confirmed that MUC1/A is associated with alleles containing the large size class of tandem repeats. Therefore, we propose that one factor in susceptibility to dry eye disease may be the lengths of the MUC1 VNTR in conjunctival epithelium based on the rationale that longer VNTR provide better lubrication and greater protection of the ocular surface against inflammation.

Alternative Splicing↗

PubMed2006

Expression in human ocular surface tissues of the GalNAc-transferases that initiate mucin-type O-glycosylation.

PURPOSE: Mucins are highly glycosylated proteins that act as lubricants, protectants, and mediators of signal transduction. The UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family members initiate mucin-type O-glycosylation. Because O-glycosylation provides mucins with the viscoelastic properties required for proper mucin function, ppGaNTase expression is vital to the maintenance of healthy epithelial surfaces including the ocular surface. Differences of expression of ppGaNTase isoforms might be factors influencing susceptibility to dry eye disease. Therefore, we determined the expression of the ppGaNTase isoforms in normal human ocular surface tissues and the conjunctival epithelium from patients with aqueous-deficient dry eye. METHODS: Expression of ppGaNTase isoforms was quantitated by real-time reverse transcriptase polymerase chain rection (RT-PCR). RESULTS: Conjunctiva and cornea expressed multiple ppGaNTase isoforms, with isoforms T12 and T4 being the most strongly expressed in conjunctiva and T12 and T3 in the cornea. In contrast, lacrimal gland expressed fewer isoforms and had lower total ppGaNTase expression. Brush cytology was found to be superior to impression cytology for harvesting conjunctival epithelium in terms of ease of use, safety, and reproducibility of results. Similar to whole conjunctiva, the strongest isoforms in conjunctival epithelial cells were T12 and T4, followed by T3, T1, T5, and T2. No significant differences of ppGaNTase expression were found between the conjunctival epithelium of dry eye and normal control groups. CONCLUSION: Human ocular surface tissues express multiple ppGaNTase isoforms, suggesting a requirement for glycosylating diverse mucin-type substrates. However, there is no evidence to date to suggest that differences of ppGaNTase expression levels might contribute to susceptibility to dry eye disease.

Conjunctiva↗

PubMed2006

Glycoprotein 340 in normal human ocular surface tissues and tear film.

The tear film is a complex mixture of secreted fluid, ions, proteins, glycoproteins, and lipids that lubricates and protects the ocular surface. Recently, several antimicrobial peptides have been described in the tear fluid. In this study, we describe the presence of the large secreted glycoprotein gp340 in the tear film. Western blot analysis showed that gp340 is abundant in secreted tears and in the lacrimal glands. Lesser amounts of gp340 were detected in the cornea and conjunctiva. Consistent with Western blot data, reverse transcription-PCR and real-time quantitative PCR showed that gp340 transcripts were abundant in lacrimal gland tissue and were also present in the cornea and conjunctiva. Immunohistochemistry localized gp340 to the acinar cells of the lacrimal gland and the deeper layers of the conjunctival epithelium. gp340 was not detected in conjunctival goblet cells. In the cornea, gp340 was present only in a peripheral band of basal epithelial cells, suggesting that gp340 may play a role in the cycle of corneal epithelial renewal. To determine if tear film gp340 may function as a bacterial agglutinin as it does in saliva, tears were incubated with streptococcal cells and the formation of bacterial aggregates was monitored. Addition of tears to late-exponential-phase Streptococcus mutans cells resulted in time- and dose-dependent aggregation of the bacteria. Furthermore, Western blot analysis confirmed the presence of cell-associated gp340 in isolated bacterial aggregates. The ocular pathogen Staphylococcus aureus, but not Pseudomonas aeruginosa, also aggregated when incubated with tears. These results suggest that gp340 is a normal component of the tear film and that the glycoprotein may function as a bacterial agglutinin.

Adult↗

PubMed2004

An inhibitor of O-glycosylation induces apoptosis in NIH3T3 cells and developing mouse embryonic mandibular tissues.

The family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases) is responsible for initiating mucin-type O-linked glycosylation in higher eukaryotes. To begin to examine the biological role of O-linked glycosylation, mammalian cells were treated with a small molecule inhibitor (designated 1-68A, Ref. 15) of ppGaNTase activity. NIH3T3 cells exposed to the inhibitor were shown to undergo a significant reduction in cell surface O-glycosylation as detected by staining with jacalin and peanut agglutinin lectins after 30 min of treatment; no reduction in staining using antibodies to O-linked N-acetylglucosamine or the lectin concanavalin A was detected. Apoptosis was also observed in treated cells after 45 min of exposure, ostensibly following the O-glycosylation reduction. Overexpression of several different ppGaNTase isoforms restored cell surface O-glycosylation and rescued inhibitor-induced apoptosis. Additionally, mouse embryonic mandibular organ cultures exposed to 1-68A developed abnormally, presumably because of epithelial and mesenchymal apoptosis that followed a reduction in jacalin and peanut agglutinin staining. Our studies suggest that mucin-type O-linked glycosylation may be required for normal development and that ppGaNTases may play a role in the regulation of apoptosis.

Animals↗