Substrate specificities and intracellular distributions of three N-glycan processing enzymes functioning at a key branch point in the insect N-glycosylation pathway

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.

FIGURE 1.

A key N-glycosylation pathway branch point. The initial steps in the insect and mammalian cell N-glycosylation pathways are similar or identical and produce the same N-glycan processing intermediate, MGn (boxed). GNT-I contributes to MGn production by adding N-acetylglucosamine to the mannose residue on the α3 branch of the M5 precursor, as shown. In insect cells, MGn is typically trimmed by FDL, which yields paucimannosidic N-glycan products. In mammalian cells, MGn is elongated by GNT-II and other glycosyltransferases, which yield hybrid and complex N-glycan products. However, recent studies have shown that insect cells also have the biochemical machinery required to produce hybrid and complex, as well as paucimannosidic N-glycans. Thus, insect cells have a branched N-glycosylation pathway in which MGn is the key branch point intermediate for alternative processing and production of structurally distinct N-glycans. Previous studies suggest that GNT-I, FDL, and GNT-II are major factors determining the outcome of this branched pathway.

FIGURE 2.

Expression and purification of recombinant Sf-GNT-I and -II. The His₆-tagged ectodomains of Sf-GNT-I and -II were expressed in recombinant baculovirus-infected Sf cells and purified from the extracellular fraction by nickel affinity chromatography, as described under “Experimental Procedures.” Samples of the purified proteins were treated with buffer alone (−) or PNGase-F (+), and then equal amounts of the untreated or treated proteins were analyzed by SDS-PAGE with Coomassie Brilliant Blue staining (CBB, left panel) or immunoblotting with an antiserum specific for His₆ (α-6xHis, right panel). The calculated molecular masses of the recombinant His₆-tagged Sf-GNT-I and -II ectodomains minus the signal peptides are 49.4 and 57.1 kDa, respectively.

FIGURE 3.

Enzymatic characterization of recombinant Sf-GNT-I and -II. The pH and metal requirements of Sf-GNT-I and -II were determined using an HPLC assay with MM-PA and MGn-PA as the acceptor substrates, respectively, as described under “Experimental Procedures.” Sf-GNT-I (A) and -II (B) activities at various pH values are expressed as percentages of the optima. Sf-GNT-I (C) and -II (D) activities in the presence of various metals are expressed as a percentage of the levels observed in the presence of Mn²⁺ (100%).

FIGURE 4.

Substrate specificities of recombinant Sf-GNT-I and -II. Purified recombinant Sf-GNT-I was incubated with M5-PA (A), MM-PA (B), or GnM-PA (C) in the presence of 10 mm Co²⁺ and 1 mm UDP-GlcNAc at pH 7.5. Purified recombinant Sf-GNT-II was incubated with MGn-PA (D and E) or MM-PA (F) in the presence of 10 mm Mn²⁺ and 1 mm UDP-GlcNAc at pH 6.7. In E, Sf-GNT-II was incubated with MGn-PA as in D, except the incubation time was extended 100-fold. Each panel shows the RP-HPLC profile obtained with the indicated reaction products.

FIGURE 5.

Intracellular distributions of GFP- or RFP-tagged Sf gene products. Sf cells were co-transfected with plasmids encoding various C-terminal GFP- or RFP-tagged Sf N-glycan processing enzymes, and then phase-contrast and fluorescence images were taken, as described under “Experimental Procedures.” GFP-tagged and RFP-tagged Sf-GNT-I (A), GFP-tagged and RFP-tagged Sf-GNT-II (B), GFP-tagged Sf-GNT-I and RFP-tagged Sf-GNT-II (C), GFP-tagged Sf-GNT-II and RFP-tagged Sf-GNT-I (D), GFP-tagged SfMan-I and RFP-tagged Sf-GNT-I (E), GFP-tagged Sf-Man-I and RFP-tagged Sf-GNT-II (F), GFP-tagged Sf-FDL and RFP-tagged Sf-GNT-I (G), and GFP-tagged Sf-FDL and RFP-tagged Sf-GNT-II (H) are shown. Phase-contrast images are shown in the left-hand column, GFP fluorescence in the second column, RFP fluorescence in the third column, merged GFP and RFP fluorescence pattern in the fourth column, and merged fluorescence patterns overlaid on phase-contrast images are shown in the right-hand column.

FIGURE 6.

Intracellular distribution of GFP-tagged Sf-FDL and various organelles. Sf cells were transfected with a plasmid encoding GFP-tagged Sf-FDL and either co-transfected with plasmids encoding RFP-tagged marker proteins or otherwise labeled with red fluorescent markers, and then phase-contrast and fluorescence images were taken, as described under “Experimental Procedures.” Cell surface (A), nuclei (B), late endosomes/multivesicular bodies (C), lysosomes (D), ER (E), and Golgi (F) are shown. The columns are as described in the legend to Fig. 5.

FIGURE 7.

Intracellular distribution of GFP-tagged Dm-FDL and RFP-tagged Golgi markers. Sf9 or S2 cells were co-transfected with plasmids encoding GFP-tagged Dm-FDL and either RFP-tagged Sf-GNT-I or RFP-tagged Dm-ΔGMII, and then phase-contrast and fluorescence images were taken, as described under “Experimental Procedures.” GFP-tagged Dm-FDL and RFP-tagged Sf-GNT-I in Sf9 cells (A), GFP-tagged Dm-FDL and RFP-tagged Sf-GNT-I in S2 cells (B), GFP-tagged Dm-FDL and RFP-tagged Dm-ΔGMII in S2 cells (C) are shown. The columns are as described in the legend to Fig. 5.