Substrate Specificities and Intracellular Distributions of Three N-glycan Processing Enzymes Functioning at a Key Branch Point in the Insect N-Glycosylation Pathway

Christoph Geisler,Donald L Jarvis

doi:10.1074/jbc.m111.296814

Abstract

Man(α1-6)[GlcNAc(β1-2)Man(α1-3)]ManGlcNAc(2) is a key branch point intermediate in the insect N-glycosylation pathway because it can be either trimmed by a processing β-N-acetylglucosaminidase (FDL) to produce paucimannosidic N-glycans or elongated by N-acetylglucosaminyltransferase II (GNT-II) to produce complex N-glycans. N-acetylglucosaminyltransferase I (GNT-I) contributes to branch point intermediate production and can potentially reverse the FDL trimming reaction. However, there has been no concerted effort to evaluate the relationships among these three enzymes in any single insect system. Hence, we extended our previous studies on Spodoptera frugiperda (Sf) FDL to include GNT-I and -II. Sf-GNT-I and -II cDNAs were isolated, the predicted protein sequences were analyzed, and both gene products were expressed and their acceptor substrate specificities and intracellular localizations were determined. Sf-GNT-I transferred N-acetylglucosamine to Man(5)GlcNAc(2), Man(3)GlcNAc(2), and GlcNAc(β1-2)Man(α1-6)[Man(α1-3)]ManGlcNAc(2), demonstrating its role in branch point intermediate production and its ability to reverse FDL trimming. Sf-GNT-II only transferred N-acetylglucosamine to Man(α1-6)[GlcNAc(β1-2)Man(α1-3)]ManGlcNAc(2), demonstrating that it initiates complex N-glycan production, but cannot use Man(3)GlcNAc(2) to produce hybrid or complex structures. Fluorescently tagged Sf-GNT-I and -II co-localized with an endogenous Sf Golgi marker and Sf-FDL co-localized with Sf-GNT-I and -II, indicating that all three enzymes are Golgi resident proteins. Unexpectedly, fluorescently tagged Drosophila melanogaster FDL also co-localized with Sf-GNT-I and an endogenous Drosophila Golgi marker, indicating that it is a Golgi resident enzyme in insect cells. Thus, the substrate specificities and physical juxtapositioning of GNT-I, GNT-II, and FDL support the idea that these enzymes function at the N-glycan processing branch point and are major factors determining the net outcome of the insect cell N-glycosylation pathway.

Highlights

Insect cells have a branched protein N-glycosylation pathway
The roles of some of these enzymes in the insect N-glycosylation pathway had been surmised from the substrate specificities observed in previous assays of crude microsomal fractions isolated from Spodoptera frugiperda (Sf) cells [3, 11] and, in the case of glycans. N-Acetylglucosaminyltransferase I (GNT-I) and -II, from their established roles in the mammalian pathway [1]
It was likely that insect GNT-I was involved in the production of MGn, which was subsequently trimmed by fused lobes (FDL) or, at a much lower frequency, elongated by insect glycans or elongated by N-acetylglucosaminyltransferase II (GNT-II)

Summary

Introduction

Insect cells have a branched protein N-glycosylation pathway. Results: Substrate specificities and intracellular distributions of three insect N-glycan processing enzymes were determined. Conclusion: Sf-GNT-I, Sf-GNT-II, and Sf-FDL function around the branch point and determine the net outcome of the insect protein N-glycosylation pathway. Man(␣1– 6)[GlcNAc(␤1–2)Man(␣1–3)]ManGlcNAc2 is a key branch point intermediate in the insect N-glycosylation pathway because it can be either trimmed by a processing ␤-Nacetylglucosaminidase (FDL) to produce paucimannosidic N-glycans or elongated by N-acetylglucosaminyltransferase II (GNT-II) to produce complex N-glycans. Fluorescently tagged Drosophila melanogaster FDL co-localized with Sf-GNT-I and an endogenous Drosophila Golgi marker, indicating that it is a Golgi resident enzyme in insect cells. The substrate specificities and physical juxtapositioning of GNT-I, GNT-II, and FDL support the idea that these enzymes function at the N-glycan processing branch point and are major factors determining the net outcome of the insect cell N-glycosylation pathway

Objectives

Results

Conclusion