DIFFERENTIATION-DEPENDENT EXPRESSION OF ALPHA-2,3-SIALYLTRANSFERASE IN RABBIT CORNEAL EPITHELIUM

PURPOSE. Lectin studies have shown that in the rabbit corneal epitheli um, alpha-2,3 sialylation of O-linked glycans differentiates limbal an d corneal epithelial cell phenotypes. Because sialic acid can be regul ated at the level of the expression of sialyltransferases (STs), the p urpose of the present study was to analyze the expression of alpha-2,3 STs in this tissue. METHODS. Reverse transcription-polymerase chain re action (RT-PCR) was used to generate ST cDNA from total rabbit corneal epithelium RNA using primers selected from the sequences of three pre viously cloned STs capable of catalyzing the transfer of sialic acid t o O-linked oligosaccharides, human placental Gal beta-1,3GalNAc-Gal be ta-1,4GluNAc alpha-2,3ST (STZ), and mouse brain Gal beta-1,3GalNAc alp ha-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 syn thesized and used to prepare an anti-ST goat antiserum. The molecular weights of immunodetectable polypeptides and their distribution in cry ostat sections of the limbocorneal area were investigated by western b lot analysis and indirect immunofluorescence, respectively. RESULTS. R T-PCR yielded cDNA of expected basepair length for STZ and ST3Gal II. The rabbit STZ cDNA was 86% identical with its human equivalent. Its m RNA was confined to the cornea, mainly in basal epithelial cells, and was not expressed in the limbus. Western blot analysis identified a ba nd at 37 kDa whose binding was abolished by preincubation of the antis erum with the immunization peptide. Immunohistologic analysis revealed the presence of immunoreactive epitopes in all basal cells of the cor nea but not in the limbus. CONCLUSIONS. STZ mRNA and the enzyme itself are expressed in the basal layer of the corneal epithelium bur are ab sent in the limbus. This enzyme's de novo expression seems thus respon sible for the differential expression of alpha-2,3 sialylation along t he limbocorneal differentiation axes. At least one more alpha-2,3ST is also present in the epithelium.