Biosynthese und Funktionen der terminalen Glukuronsäure im Glukuronyl-T- und HNK-1-Antigen von Drosophila melanogaster
Final Report Abstract
The Drosophila glucuronyltransferase GlcAT-I is highly homologues to the human transferase GlcAT-I. Both were shown to be specific for the addition of glucuronic acid (GlcA) to the core region of glycosaminoglycan (GAG) chains. They exhibit no activity towards the glycan chains on glycoproteins or glycolipids. Mammalian GlcAT-P and GlcAT-S, on the other hand, have rigid substrate specificities towards the Gal 1-4GlcNAc epitope, and hence are involved in formation of the HNK1-epitope on glycoproteins and glycolipids. While the expression of these enzymes in mammals is limited to neuronal tissue and kidney, the Drosophila orthologues are expressed ubiquitously. Moreover, dGlcAT-S and dGlcAT-P are expressed during all developmental stages of the fly. In vitro, the enzymes exhibit broad substrate specificities with formation of N-linked HNK-1 and O-linked glucuronyl-T antigens. This study was undertaken to learn in depth about the substrate specificities of the Drosophila GlcAT isoenzymes P and S in vivo and in vitro. To investigate the ß1-3-glucuronyltransferase isoforms in vivo und in vitro, we generated vectors for recombinant expression of dGlcAT-P and dGlcAT-S in cultivated Drosophila Schneider 2 (S2) cells. Moreover, the complete cDNAs of dGlcAT-P and dGlcAT-S were cloned into the mammalian expression vector pCDNA3.1 for recombinant expression in CHO-Lec2 cells. These mutant CHO cells lack sialic acid on their N- and O-linked glycans. As a consequence, these cells expose acceptors for glucuronyltransferases on glycoproteins and glycolipids making them a suitable cell model free of endogenous enzyme activities. Besides mass spectrometry of glycans, two monoclonal antibodies formed the basis for a structural discrimination of the enzymatic products. MAb M6749 was found to be specific for the HNK1-epitope, while mAb 114-2G11-A generally detects terminal 1-3-linked glucuronic acid modifications. Overexpression in S2 cells of dGlcAT-P led to a higher expression of the O-linked glucuronyl-T antigen in vivo, while overexpression of dGlcAT-S did not induce significant changes in the fraction of mucin-type O-glycans. In the transfected Drosophila S2-cells most of the glycoprotein-modifying glucuronic acid was shown to be bound to O-glycan chains, while only minor amounts are linked to N-glycans. In this study we show also that the Drosophila enzymes dGlcAT-S and dGlcAT-P can transfer GlcA in vitro to both, the O-linked T-antigen as well as to N-linked LacNAc-chains. In vivo glucuronylation studies in GlcAT-P transfected CHO-Lec2 cells revealed modification of both, N- and O-linked chains on cotransfected nidogen-1, whereas only N-glycans were modified by the GlcAT-S isoenzyme. Immunostaining of western blots with mAb 114-2G11-A (anti-GlcA) of cell lysates from S2-wt and dGlcAT-P (P) or dGlcAT-S (S) overexpressing cells revealed a strong increase of GlcA-epitopes in cells overexpressing GlcAT-P in the mass range around 70 kDa. These proteins were identified by mass spectrometric proteomics as belonging to a group of chaperones. In conclusion, only dGlcAT-P accepts the O-linked disaccharide as substrate, while both isoenzymes (P, S) are able to synthesize the non-sulfated HNK1-epitope on N-glycans. As previously speculated for the role of GlcAT-S in mouse, Drosophila GlcAT-S may be active in Drosophila also on targets other than glycoproteins. The study revealed insight into the substrate specificities of two fly enzymes involved in the formation of the important developmental HNK1 antigen and provided evidence that a group of chaperones in Drosophila cells might be modified by glucuronylated glycans.
Publications
- Functional Analysis of the Glucuronyltransferases GlcAT-P and GlcAT-S of Drosophila melanogaster: Distinct Activities towards the O-linked T-antigen. Biomolecules. 2016; 6(1):8
Breloy I, Schwientek T, Althoff D, Holz M, Koppen T, Krupa A, Hanisch FG
(See online at https://doi.org/10.3390/biom6010008)
