Molecular mechanisms for sorting lysosomal proteins

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Project Summary The objective of this proposal is to gain mechanistic and pathological understanding of human GlcNAc-1- phosphotransferase (PTase), an enzyme which modifies lysosomal hydrolases. Lysosomes contain several dozen acid hydrolases that break down unwanted proteins and lipids. Lysosomal enzymes acquire a mannose- 6-P moiety in the Golgi apparatus by the action of PTase, which uses UDP-GlcNAc as a donor substrate. The mannose-6-P functions like a zip code that guides the delivery of these enzymes to the lysosomes. PTase is encoded by the GNPTAB and GNPTG genes and assembles a 340-kDa heterohexamer of α2β2γ2. PTase selectively modifies lysosomal (but not non-lysosomal) glycoproteins transiting the ER–Golgi system. It has been unclear how PTase catalyzes the GlcNAc-P transfer reaction and how it distinguishes lysosomal from non-lysosomal glycoproteins. Mutations in the genes encoding PTase cause mucolipidosis II and III and lead to abnormal body structure, mental retardation, and several neurological diseases. In preliminary studies, we have determined the atomic structure of the catalytic core of the dimeric α2β2 subcomplex in the apo form. We have identified an inhibitory hockey stick motif that moves in and out of the catalytic pocket in the absence of substrates. We have also derived a nm-resolution cryo-EM 3D map of the holoenzyme. Building on these strong preliminary studies, we propose to determine the structures of the catalytic core bound to the donor substrate UDP-GlcNAc and the acceptor substrate mimic α-methylmannoside, and to perform structure-guided mutagenesis and in vitro activity assays. These structure-function studies of the catalytic core will enable us to formulate the unique phospho-glycosyl transfer reaction mechanism. We also propose to carry out systematic mutational and functional assays to examine the many reported disease-causing mutations. Finally, we propose to study the structures of a large lysosomal hydrolase-binding peripheral region and the γ-subunit of the PTase holoenzyme and to explore how they interacts with selected substrate lysosomal hydrolases. The proposed research will address the molecular mechanism of a key signaling pathay in glycobiology and will provide molecular insights into how mutations in the PTase cause the associated human diseases.