Structural And Functional Analysis Of N-Acetylglucosamine-1- Phosphotransferase And β- Hexosaminidase A

Abstract

To gain a better insight into the complex process of sorting and delivery of lysosomal enzymes into lysosomes, in this thesis, the focus was on understanding the structure-function relationships of GlcNAc-1-phosphotransferase, the enzyme responsible for tagging the soluble lysosomal enzymes with mannose-6-phosphate (M6P), and also on β-hexosaminidase A, an enzyme that utilizes M6P receptor dependent pathway of delivery to lysosomes, and responsible for degradation of GM2 ganglioside. Mucolipidosis II and III, lysosomal storage disorders result from mutations in GNPTAB and GNPTG genes encoding the αβ- and γ-subunits of the G1cNAc-1-phosphotransferase, respectively. In Chapter 2, four MLII and three MLIII cell lines were screened for the presence of mutations and 22 novel genetic variants in GNPTAB gene and 2 previously reported mutations (one in GNPTAB and another in GNPTG) including several potential polymorphic sites in GNPTAB gene were identified. In addition, it was also shown that mutations in one subunit affect the assembly and intracellular distribution of the other subunits. Further, phenotypic correction of defect of MLII and MLIIIC fibroblasts was demonstrated by in vitro transfection with wild type cDNAs encoding the α/β- and γ-subunits of G1cNAc-1-phosphotransferase, respectively.

Primary sequences of soluble lysosomal enzymes have not revealed any common targeting motif for their recognition by G1cNAc-1-phosphotransferase. Since the recognition site resides in the γ-subunit of G1cNAc-1-phosphotransferase (Protein id: NP_115909), structural studies were undertaken to better understand how the enzyme recognizes its substrates. Accordingly in Chapter 3, a number of physical characteristics of the expressed, in vitro folded γ-subunit, a 305 amino acid soluble glycoprotein responsible for substrate recognition were studied. The main focus of Chapter 4 was on β-hexosaminidase A that requires GM2 activator protein to hydrolyze the GM2 ganglioside substrate. The studies of refinement of the molecular model created for the interaction of β-hexosaminidase A and its substrate-specific cofactor, GM2 activator protein, indicated the necessity of the α-loop (α280GSEP 283) for functional interaction between β-hexosaminidase A and GM2 activator proteins in hydrolyzing GM2 ganglioside. In conclusion, this thesis enriches the current view of structure-function analysis of the proteins responsible for lysosomal trafficking and degradation.