GlcNAc-1-phosphotransferase: Structure and Lysosomal Hydrolase Recognition

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A) Rationale/Aim: The Golgi-resident enzyme, UDP-N-acetylglucosamine-1-phosphotransferase (GNPT) initiates the addition of the mannose-6-phosphate (M6P) moiety to the N-glycans of soluble lysosomal hydrolases. This modification is required for the targeting of these enzyme to lysosomes but how GNPT recognizes its structurally diverse substrates is unknown. The short-term goal of this work (the subject of this proposal) is to determine the structure of GNPT and insight into the basis for its substrate specificity. In the long-term we seek to fully describe the basis for GNPT's ability to specifically recognize its over 50 lysosomal enzyme substrates. ******B) Background: GNPT is a ~360 kDa complex composed of three subunits (222). Each of these subunits contain multiple domains that play different roles in binding and catalysis. Among these, the so-called stealth domain (a sugar-phosphate transferase domain in some bacteria) forms the catalytic core of GNPT and the N1, N2 and DMAP protein interaction domains are involved in substrate recognition. Notably, the subunit is not required for the recognition of some lysosomal enzymes and the protein interaction domains play different roles in the recognition of different substrates. ******C) Preliminary Results: We have expressed the human GNPTAB (the and subunits are generated by proteolysis) and GNPTG cDNA's both separately and in combination using our PiggyBac-based mammalian cell expression system. Various forms of the enzyme have been characterized by negative strain EM and our preliminary cryo-EM analysis of one of them establishes that it can be successfully frozen in thin ice. The resulting 2D class averages from the cryo data provide evidence of 2-fold symmetry and a sample suitable for high-resolution structure determination. We have also expressed several lysosomal enzymes to be used in characterizing the specificity of GNPT.******D) Approach: Single particle cryo-EM analysis will be used to determine the structure of various forms of GNPT. In addition, x-ray crystal structures of GNPT domains/fragments will be determined to add high resolution details. Key among the x-ray structures will be an active form of GNPT lacking the protein interaction domains and the entire subunit and two homologous bacterial stealth domains. In addition, biochemical analysis will be used to determine the role played by various GNPT domains/subunits in the recognition of selected lysosomal enzyme substrates. ******E) Outcomes: This work will lead to the first structure of GNPT and/or fragments of it including the catalytic core or stealth domain conserved across evolution. GNPT uses different domains to bind different lysosomal enzymes and the work will also provide insight into the structural and biochemical basis for that process.**