Analysis of the biogenesis of heparan sulfate acetyl-CoA:alpha-glucosaminide N-acetyltransferase provides insights into the mechanism underlying its complete deficiency in mucopolysaccharidosis IIIC

Abstract

Heparan sulfate acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT) catalyzes the transmembrane acetylation of heparan sulfate in lysosomes required for its further catabolism. Inherited deficiency of HGSNAT in humans results in lysosomal storage of heparan sulfate and causes the severe neurodegenerative disease, mucopolysaccharidosis IIIC (MPS IIIC). Previously we have cloned the HGSNAT gene, identified molecular defects in MPS IIIC patients, and found that all missense mutations prevented normal folding and trafficking of the enzyme. Therefore characterization of HGSNAT biogenesis and intracellular trafficking became of central importance for understanding the molecular mechanism underlying the disease and developing future therapies. In the current study we show that HGSNAT is synthesized as a catalytically inactive 77-kDa precursor that is transported to the lysosomes via an adaptor protein-mediated pathway that involves conserved tyrosine- and dileucine-based lysosomal targeting signals in its C-terminal cytoplasmic domain with a contribution from a dileucine-based signal in the N-terminal cytoplasmic loop. In the lysosome, the precursor is cleaved into a 29-kDa N-terminal α-chain and a 48-kDa C-terminal β-chain, and assembled into active ∼440-kDa oligomers. The subunits are held together by disulfide bonds between at least two cysteine residues (Cys(123) and Cys(434)) in the lysosomal luminal loops of the enzyme. We speculate that proteolytic cleavage allows the nucleophile residue, His(269), in the active site to access the substrate acetyl-CoA in the cytoplasm, for further transfer of the acetyl group to the terminal glucosamine on heparan sulfate. Altogether our results identify intralysosomal oligomerization and proteolytic cleavage as two steps crucial for functional activation of HGSNAT.

FIGURE 1.

Comparison of recombinant proteins expressed from the two predicted ATG start sites of HGSNAT. COS-7 cells were transfected with HGSNAT-TAP plasmids and assayed for N-acetyltransferase activity, analyzed by Western blot using anti-CBP antibody or labeled with [³⁵S]Met/Cys. A, N-acetyltransferase activity in the cell homogenates shown relative to the wild type HGSNAT-TAP-long (mean of three independent experiments; error bars represent SD of three independent experiments). B, representative Western blot of cell homogenates. C, synthesis and processing of HGSNAT precursors: the cells were labeled for 15 min with [³⁵S]Met/Cys, and chased for the indicated time; HGSNAT-TAP was affinity purified prior to analysis on SDS-PAGE and exposure to autoradiographic film. L, long; S, short; M, M29A.

FIGURE 2.

Metabolic labeling of HGSNAT. Six hours post-transfection with pCTAP-HGSNAT plasmid, cells were incubated for 24 h with 50 mm NH₄Cl and then for the indicated time in a normal medium containing 7 μm cycloheximide to inhibit de novo protein synthesis. A, N-acetyltransferase activity in the cell homogenates (mean of three independent experiments; error bars represent SD of three independent experiments). B, Western blot of cell homogenates using anti-CBP antibody. C, cells were labeled for 1 h with [³⁵S]Met/Cys, and chased in normal medium for the indicated time; HGSNAT-TAP was affinity purified prior to analysis by SDS-PAGE and exposure to autoradiographic film. NT, non-transfected cells.

FIGURE 3.

Identification of cysteine residues involved in HGSNAT oligomerization. COS-7 cells were transfected with HGSNAT-TAP plasmid constructs encoding mutations of cysteine residues. A, N-acetyltransferase activity in cell homogenates shown relative to the wild type (mean of three independent experiments; error bars represent SD of three independent experiments). B and C, representative Western blots of cell homogenates using anti-CBP antibody in non-reducing and reducing conditions, respectively. NT, non-transfected cells.

FIGURE 4.

Identification of HGSNAT regions important for lysosomal targeting. COS-7 cells were transfected with HGSNAT-TAP plasmids bearing mutations in the predicted targeting signals and studied by immunohistochemistry and confocal microscopy or analyzed for N-acetyltransferase activity and by Western blot. A, mutations introduced in HGSNAT sequence. B, transfected cells were fixed and stained with mouse monoclonal anti-LAMP-2 antibodies (red) and rabbit polyclonal anti-CBP antibodies (green). Slides were studied on a Zeiss LSM510 inverted confocal microscope. Magnification ×630. C, N-acetyltransferase activity in cell homogenates shown relative to the wild type (mean of three or four independent experiments; error bars represent SD). D, representative Western blots of cell homogenates. NT, non-transfected cells.

FIGURE 5.

Identification of the histidine acceptor residue in the HGSNAT active site. COS-7 cells were transfected with HGSNAT-TAP plasmids encoding mutations of histidine residues and analyzed for N-acetyltransferase activity and by Western blot. Some mutants were expressed in human fibroblasts for immunohistochemical analysis. A, N-acetyltransferase activity in the cell homogenates shown relative to the wild type (mean of two independent experiments; error bars represent SD; NT, non-transfected cells). B, representative Western blots of cell homogenates. C, transfected human skin fibroblasts were fixed and stained with rabbit polyclonal anti-CBP antibodies (green) and mouse monoclonal anti-LAMP-2 or mouse monoclonal anti-calnexin antibodies (red). Slides were studied on a Zeiss LSM510 inverted confocal microscope. Magnification ×630. D, wild type and H269A-transfected COS-7 cell homogenates labeled with [¹⁴C]acetyl-CoA were separated by SDS-PAGE (1.65 μCi per lane), lanes were cut into 23 bands and analyzed using a scintillation counter.

FIGURE 6.

Oligomeric composition of HGSNAT active form. COS-7 cells were transfected with HGSNAT-TAP-short (WT), del624–635, or H269A plasmids. Protein extracts were analyzed by size exclusion chromatography on a FPLC Superdex 200 column or by PFO-PAGE. A, N-acetyltransferase activity in the elution fractions from the Superdex 200 column loaded with wild type (WT) enzyme. Elution volumes of protein size standards are indicated above the graph: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (240 kDa), aldolase (158 kDa), and chymotrypsinogen A (25 kDa). B, elution fractions were analyzed by Western blot using anti-CBP antibody, in reducing (+DTT) or non-reducing (−DTT) conditions for the WT and reducing conditions for the mutants. C, cell homogenates were suspended in PFO-PAGE sample buffer, separated on 7% PFO-PAGE, and analyzed by Western blot using anti-CBP antibody. Positions of the commercial molecular mass markers (260, 160, and 110 kDa), thyroglobulin (669 kDa), and ferritin (440 kDa) are indicated.