Compensatory expression of human N-Acetylglucosaminyl-1-phosphotransferase subunits in mucolipidosis type III gamma

Abstract

The N-Acetylglucosaminyl-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate (M6P) recognition markersessential for efficient transport of lysosomal hydrolases to lysosomes. The phosphotransferase is composed of six subunits (alpha2, beta2, gamma2). The alpha- and beta-subunits are catalytically active and encoded by a single gene, GNPTAB, whereas the gamma-subunit encoded by GNPTG is proposed to recognize conformational structures common to lysosomal enzymes. Defects in GNPTG cause mucolipidosis type III gamma, which is characterized by missorting and cellular loss of lysosomal enzymes leading to lysosomal accumulation of storage material. Using plasmon resonance spectrometry, we showed that recombinant gamma-subunit failed to bind the lysosomal enzyme arylsulfatase A. Additionally, the overexpression of the gamma-subunit in COS7 cells did not result in hypersecretion of newly synthesized lysosomal enzymes expected for competition for binding sites of the endogenous phosphotransferase complex. Analysis of fibroblasts exhibiting a novel mutation in GNPTG (c.619insT, p.K207IfsX7) revealed that the expression of GNPTAB was increased whereas in gamma-subunit overexpressing cells the GNPTAB mRNA was reduced. The data suggest that the gamma-subunit is important for the balance of phosphotransferase subunits rather for general binding of lysosomal enzymes.

Enzyme-specific differences in mannose phosphorylation between GlcNAc-1-phosphotransferase αβ and γ subunit deficient zebrafish support cathepsin proteases as early mediators of mucolipidosis pathology

“…These data indicate that loss of the γ subunit does not reduce phosphotransferase activity towards a simple sugar substrate. Further, RT-PCR analyses of gnptg −/− embryos showed normal levels of gnptab mRNA, suggesting that the γ subunit does not influence αβ expression in this system, in contrast to what has been noted in cells (26,27). …”

Proteolytic Processing of the γ-Subunit Is Associated with the Failure to Form GlcNAc-1-phosphotransferase Complexes and Mannose 6-Phosphate Residues on Lysosomal Enzymes in Human Macrophages

“…Furthermore, quantitative real-time PCR showed that the relative mRNA level of both GNPTAB and GNPTG were elevated to a similar extent compared with those in human fibroblasts or Hela cells (Table 1), indicating the stability of mRNAs. In contrast, a characteristic finding in fibroblasts of MLIII patients exhibiting mutations in GNPTG gene, is the compensatory increase in GNPTAB mRNA expression (11,24), which was not observed in macrophages. These data support the idea that neither genomic nor transcriptional alterations in GNPTAB/GNPTG are responsible for the loss in GlcNAc-1-phosphotransferase activity in macrophages.…”

“…The construct was sequenced on an ABI PRISM 377 DNA Sequencer (University Medical Center Service Laboratory, Hamburg-Eppendorf). The expression vector coding for the N-terminally Myc-tagged ␥-subunit has been described elsewhere (11).…”

“…Western Blot Analysis-The expression of the ␥-subunit of the GlcNAc-1-phosphotransferase in cell extracts or fractions after size exclusion chromatography was analyzed by Western blotting as described recently (11).…”

A Novel Xylosylphosphotransferase Activity Discovered in Cryptococcus neoformans

“…The UDP-GlcNAc phosphotransferase, which has an ␣ 2 ␤ 2 ␥ 2 subunit structure, catalyzes the initial step in the mannose-6-phosphate modification of lysosomal hydrolases that is necessary for targeting these proteins to the lysosome. The ␣ and ␤ subunits are encoded by a single gene and are thought to contain the catalytic portion of the protein (39), whereas the ␥ subunit is encoded by a separate gene and is thought be a regulatory element (40,41). Based on our studies, Xpt1p cannot utilize FIGURE 9.…”