Abstract
The distinction between the different classes of glycolipids is conditioned by the action of specific core transferases. The entry point for lacto-series glycolipids is catalyzed by the beta1,3 N-acetylglucosaminyltransferase GlcNAc(beta1,3)Gal(beta1,4)Glc-ceramide (Lc3) synthase enzyme. The Lc3 synthase activity has been shown to be regulated during development, especially during brain morphogenesis. Here, we report the molecular cloning of a mouse gene encoding an Lc3 synthase enzyme. The mouse cDNA included an open reading frame of 1131 base pairs encoding a protein of 376 amino acids. The Lc3 synthase protein shared several structural motifs previously identified in the members of the beta1,3 glycosyltransferase superfamily. The Lc3 synthase enzyme efficiently utilized the lactosyl ceramide glycolipid acceptor. The identity of the reaction products of Lc3 synthase-transfected CHOP2/1 cells was confirmed by thin-layer chromatography immunostaining using antibodies TE-8 and 1B2 that recognize Lc3 and Gal(beta1,4)GlcNAc(beta1,3)Gal(beta1,4)Glc-ceramide (nLc4) structures, respectively. In addition to the initiating activity for lacto-chain synthesis, the Lc3 synthase could extend the terminal N-acetyllactosamine unit of nLc4 and also had a broad specificity for gangliosides GA1, GM1, and GD1b to generate neolacto-ganglio hybrid structures. The mouse Lc3 synthase gene was mainly expressed during embryonic development. In situ hybridization analysis revealed that that the Lc3 synthase was expressed in most tissues at embryonic day 11 with elevated expression in the developing central nervous system. Postnatally, the expression was restricted to splenic B-cells, the placenta, and cerebellar Purkinje cells where it colocalized with HNK-1 reactivity. These data support a key role for the Lc3 synthase in regulating neolacto-series glycolipid synthesis during embryonic development.
Highlights
Glycolipids are ubiquitous constituents of cellular membranes
Using in situ hybridization, we provide evidence that the Lc3 synthase gene is broadly expressed in the nervous system and other tissues during mouse embryonic development but is restricted to some cell types in adult
Several structural motifs are shared by the superfamily of 1,3 glycosyltransferases including 1,3 galactosyl- [21, 26], 1,3 N-acetylglucosaminyl- [23, 27], and 1,3 N-acetylgalactosaminyltransferases [28]
Summary
Cloning and Expression of the Lc3 Synthase Gene—The human expressed sequence tag fragment AI589087 was retrieved from the EST division of GenBankTM as it showed significant similarity to the 1,3 galactosyltransferase-I, -II, -III, and -V [21, 22] and 1,3 N-acetylglucosaminyltransferase-I [23] proteins. Thin-layer Chromatography Immunostaining of Lc3 Synthase-transfected Cells—The full-length coding region of the mouse and human Lc3 synthase gene were excised from pFastbac by BamHI and XbaI digestion and subcloned into pcDNA3 for transient transfection of CHOP2/1 cells. This Chinese hamster ovary glycosylation mutant is defective in Golgi sialic acid transport and in the Mgat gene required for initiation of N-linked glycan chain synthesis [24]. In Situ Hybridization—Digoxigenin (DIG)-labeled sense and antisense riboprobes were synthesized using a DIG RNA synthesis kit (Roche Molecular Biochemicals) from a 0.7-kbp HindIII-BglII fragment of the mouse Lc3 synthase cDNA subcloned into the plasmid pGEM3Zf(ϩ) (Promega) following the instructions of the manufacturer. UDP-GalNAcc pmol/min/mg prot added a Lysate of Sf9 cells infected with a wild-type baculovirus. bLysate of Sf9 cells infected with recombinant baculovirus. c Carbohydrate donor. d (octyl), O(CH2)8CO2Me. e Lac-N-tetraose, Gal(1–3)GlcNAc(1–3)Gal(1– 4)Glc; Lac-N-neotetraose, Gal(1– 4)GlcNAc(1–3)Gal(1– 4)Glc
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
