Functions of the α, β, and γ Subunits of UDP-GlcNAc:Lysosomal Enzyme N-Acetylglucosamine-1-phosphotransferase

Yi Qian,Intaek Lee,Wang-Sik Lee,Meiqian Qian,Mariko Kudo,William M Canfield,Peter Lobel,Stuart Kornfeld

doi:10.1074/jbc.m109.068650

Abstract

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an alpha(2)beta(2)gamma(2) hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Using a multifaceted approach, including analysis of acid hydrolase phosphorylation in mice and fibroblasts lacking the gamma subunit along with kinetic studies of recombinant alpha(2)beta(2)gamma(2) and alpha(2)beta(2) forms of the transferase, we have explored the function of the alpha/beta and gamma subunits. The findings demonstrate that the alpha/beta subunits recognize the protein determinant of acid hydrolases in addition to mediating the catalytic function of the transferase. In mouse brain, the alpha/beta subunits phosphorylate about one-third of the acid hydrolases at close to wild-type levels but require the gamma subunit for optimal phosphorylation of the rest of the acid hydrolases. In addition to enhancing the activity of the alpha/beta subunits toward a subset of the acid hydrolases, the gamma subunit facilitates the addition of the second GlcNAc-P to high mannose oligosaccharides of these substrates. We postulate that the mannose 6-phosphate receptor homology domain of the gamma subunit binds and presents the high mannose glycans of the acceptor to the alpha/beta catalytic site in a favorable manner.

Highlights

Dom.wustl.edu. 3 The abbreviations used are: Man-6-P, mannose 6-phosphate; CI-MPR, cation-independent mannose 6-phosphate receptor; LC-MS/MS, liquid chromatography-mass spectrometry/mass spectrometry; DMEM, Dulbecco’s modified Eagle’s medium; Sulfo-N-hydroxysuccinimide acetate (SNA), sulfo-N-hydroxysuccinimide acetate; KO, knock out; MRH, mannose 6-phosphate receptor homology; ML, mucolipidosis
The data presented in this study provide a number of new insights into the role of the ␣/␤ and ␥ subunits of GlcNAc-1phosphotransferase
It has previously been established both by in vitro studies and analysis of mutations in patients with ML II and ML III that the ␣/␤ subunits contain the catalytic function of the transferase

Summary

EXPERIMENTAL PROCEDURES

Materials—UDP-[6-3H]GlcNAc (20 Ci/mmol) was purchased from PerkinElmer Life Sciences. [␤-32P]UDP-GlcNAc was prepared as described previously [2]. [2-3H]Mannose was purchased from MP Biomedicals (Irvine, CA). GlcNAc-1-phosphotransferase Assay—The reaction mixture contained various concentrations of the substrates and either 1.2 ␮g of the ␣2␤2␥2 or 0.97 ␮g of the ␣2␤2 GlcNAc-1-phosphotransferase in 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 75 ␮M UDP-[3H]GlcNAc (1 ␮Ci), 2 mg/ml bovine serum albumin in a final volume of 50 ␮l. Labeling of Mouse Fibroblasts—Mouse fibroblasts infected with wild-type or mutated bovine DNase I viral supernatant in 60-mm plates were labeled 4 h with 1 ml of DMEM containing 5 mM glucose, 10% dialyzed fetal calf serum, 10 mM NH4Cl, and 200 ␮Ci [2-3H]mannose and chased 4 h by addition of 300 ␮l of chase medium containing 10 mM glucose and 10 mM mannose to stop mannose uptake [31]. The immunoprecipitates eluted from protein A-Sepharose beads were treated with endoglycosidase H, and oligosaccharide analysis was carried out according to the previously described Experimental Procedures in Ref. 32

RESULTS

18 Ϯ 4 38 Ϯ 9 24 Ϯ 5 83 Ϯ 13

DISCUSSION