Purpose: Lectin studies have shown that in the rabbit corneal epithelium, alpha-2,3 sialylation of O-linked glycans differentiates limbal and corneal epithelial cell phenotypes. Because sialic acid can be regulated at the level of the expression of sialyltransferases (STs), the purpose of the present study was to analyze the expression of alpha-2,3STs in this tissue.
Methods: Reverse transcription-polymerase chain reaction (RT-PCR) was used to generate ST cDNA from total rabbit corneal epithelium RNA using primers selected from the sequences of three previously cloned STs capable of catalyzing the transfer of sialic acid to O-linked oligosaccharides, human placental Galbeta-1,3GalNAc-Galbeta-1,4GluNAcalpha-2,3ST (STZ), and mouse brain Galbeta-1,3GalNAcalpha-2,3ST types I and II (ST3Gal I and ST3Gal II). Tissue distribution of mRNA was assayed by fluorescent in situ hybridization. A synthetic peptide whose sequence was deduced from a cloned cDNA fragment was synthesized and used to prepare an anti-ST goat antiserum. The molecular weights of immunodetectable polypeptides and their distribution in cryostat sections of the limbocorneal area were investigated by western blot analysis and indirect immunofluorescence, respectively.
Results: RT-PCR yielded cDNA of expected basepair length for STZ and ST3(Gal II. The rabbit STZ cDNA was 86% identical with its human equivalent. Its mRNA was confined to the cornea, mainly in basal epithelial cells, and was not expressed in the limbus. Western blot analysis identified a band at 37 kDa whose binding was abolished by preincubation of the antiserum with the immunization peptide. Immunohistologic analysis revealed the presence of immunoreactive epitopes in all basal cells of the cornea but not in the limbus.
Conclusions: STZ mRNA and the enzyme itself are expressed in the basal layer of the corneal epithelium but are absent in the limbus. This enzyme's de novo expression seems thus responsible for the differential expression of alpha-2,3 sialylation along the limbocorneal differentiation axes. At least one more alpha-2,3ST is also present in the epithelium.