Identification of the interaction domains between α‐ and γ‐subunits of GlcNAc‐1‐phosphotransferase

Abstract

The disease-associated hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α β γ ) catalyzes the formation of mannose 6-phosphate residues on lysosomal enzymes required for efficient targeting to lysosomes. Using pull-down experiments and mutant subunits, we identified a potential loop-like region in the α-subunits comprising residues 535-588 and 645-698 involved in the binding to γ-subunits. The interaction is independent of the mannose 6-phosphate receptor homology domain but requires the N-terminal unstructured part of the γ-subunit consisting of residues 26-69. These studies provide new insights into structural requirements for the assembly of the GlcNAc-1-phosphotransferase complex, and the functions of distinct domains of the α- and γ-subunits.

Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy

“…In addition, there are four so-called spacer domains in the α/β precursor whose functions are beginning to be elucidated. Thus, we have reported that spacer-1 (residues 86–322) determines the site of cleavage of the α/β precursor and serves to limit the phosphorylation of non-lysosomal glycoproteins, 13 whereas spacer-2 contains the γ-subunit binding site 12, 14. Besides these domains, the α and β subunits also harbor four Stealth domains that together form the catalytic core of the protein.…”

The lysosomal storage disorders mucolipidosis type II, type III alpha/beta, and type III gamma: Update onGNPTABandGNPTGmutations

“…In the Golgi apparatus the α/β‐precursor is proteolytically cleaved into enzymatically active α‐ and β‐subunits by site‐1 protease (S1P; Figure a; Marschner, Kollmann, Schweizer, Braulke, & Pohl, ; Velho et al, ). The soluble γ‐subunit directly binds to the α‐subunit and enhances the GlcNAc‐1‐phosphotransferase activity for M6P modification of specific lysosomal enzymes (De Pace et al, ; Di Lorenzo et al, ; Velho, De Pace, Tidow, Braulke, & Pohl, ).…”

“…Noteworthy, by using a recombinant lysosomal enzyme instead of the artificial substrate, the GlcNAc‐1‐phosphotransferase activity has been shown to be strongly reduced, demonstrating that both Notch repeat‐like and DMAP domains are involved in the binding to lysosomal enzymes (Qian et al, ; Qian, Flanagan‐Steet, van Meel, Steet, & Kornfeld, ; van Meel et al, ). In addition, GNPTAB missense mutations in the γ‐subunit‐binding domain decrease the GlcNAc‐1‐phosphotransferase activity (Figure b), presumably by impairing the interaction between the α‐ and γ‐subunits and consequently the subunit assembly of the complex (De Pace et al, ; Velho, De Pace, et al, ). Likewise, loss‐of‐function mutations in GNPTG cause a significant reduction in enzymatic activity (De Pace et al, ; Di Lorenzo et al, ).…”

Lysosomal Proteome and Secretome Analysis Identifies Missorted Enzymes and Their Nondegraded Substrates in Mucolipidosis III Mouse Cells

“…It has been suggested that the ␥-subunits enhance the recognition and M6P formation of distinct lysosomal enzymes by the ␣and ␤-subunit (11,12), whereas other studies failed to show direct interactions of the ␥-subunits with lysosomal enzymes (6,13). Recently, we identified the ␥-subunit binding domain in a previously uncharacterized luminal region of the ␣-subunit required for maximum Gl-cNAc-1-phosphotransferase activity (14,15).…”