Viral vector-mediated delivery of competing glycosyltransferases modifies epitope expression cell specifically.

The glycoconjugate epitopes 3-fucosyl-N-acetyllactosamine (CD15) and sulfoglucuronylcarbohydrate (SGC) mediate cell adhesion events in several systems, and are regulated both spatially and temporally during cerebellar development. In cotransfection studies using COS-1 cells, competition between glycosyltransferases that utilize a common precursor involved in the final synthetic steps of these epitopes, can modulate epitope expression. For example, cotransfection of rat alpha1,3-fucosyltransferase IV (Fuc-TIV) and either rat glucuronic acid transferase P (GlcAT) or pig alpha1,3-galactosyltransferase (GalT) resulted in the dominance of either SGC or GalalphaGal epitope expression, respectively, with blockage of CD15 epitope expression.

Molecular cloning of rat alpha1,3-fucosyltransferase IX (Fuc-TIX) and comparison of the expression of Fuc-TIV and Fuc-TIX genes during rat postnatal cerebellum development.

Fucosylated glycoconjugates play an essential role in central nervous system development, but the regulation of expression of these molecules is not well understood. The final biosynthetic step for a major group of cerebellar fucosylated glycoconjugates (those bearing the developmentally regulated epitope 3-fucosyl-N-acetyllactosamine, CD15, and related fucosylated epitopes) is catalyzed by an alpha-1,3-fucosyltransferase (FucT). The major FucT activity in postnatal rat cerebellum has a specificity consistent with that encoded by either a Fuc-TIV- or Fuc-TIX-like gene, and thus the expression of these genes was investigated during postnatal rat cerebellar development.

N-glycosylation site occupancy of rat alpha-1,3-fucosyltransferase IV and the effect of glycosylation on enzymatic activity.

All mammalian alpha-1,3-fucosyltransferases (Fuc-Ts) so far characterized have potential N-glycosylation sites, but the role of these sites in enzymatic activity or localization has not been investigated. When one member of this family, rFuc-TIV, is expressed in bacteria, the unglycosylated form of rFuc-TIV has no detectable enzymatic activity. The two potential N-glycosylation sites of rFuc-TIV were mutated to determine site occupancy and the effect of site occupancy on enzyme activity and targeting of this enzyme.

Restriction of high CD15 expression to a subset of rat cerebellar astroglial cells can be overcome by transduction with adenoviral vectors expressing the rat alpha 1,3-fucosyltransferase IV gene.

Glycoconjugates bearing the epitope 3-fucosyl-N-acetyllactosamine (CD15) are believed to be involved in cell-cell interactions and are temporally and spatially regulated in the brain. In the rat postnatal cerebellum, CD15 is predominantly expressed in the molecular layer by Bergmann glial cells, but little CD15 expression is seen in other astroglia, and the basis for this restricted expression is not known. Adenoviral vectors were shown to efficiently deliver transgenes to cerebellar glial cells and were used to determine whether manipulation of glycosyltransferase activities could enhance the expression of CD15 in these cells.