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1.
Shuo Du Guopeng Wang Zhiying Zhang Chengying Ma Ning Gao Junyu Xiao 《The Journal of biological chemistry》2022,298(3)
GlcNAc-1-phosphotransferase catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels ∼60 lysosomal proteins for transport. Mutations in GlcNAc-1-phosphotransferase are known to cause lysosomal storage disorders such as mucolipidoses. However, the molecular mechanism of GlcNAc-1-phosphotransferase activity remains unclear. Mammalian GlcNAc-1-phosphotransferases are α2β2γ2 hexamers in which the core catalytic α- and β-subunits are derived from the GNPTAB (N-acetylglucosamine-1-phosphate transferase subunits alpha and beta) gene. Here, we present the cryo-electron microscopy structure of the Drosophila melanogaster GNPTAB homolog, DmGNPTAB. We identified four conserved regions located far apart in the sequence that fold into the catalytic domain, which exhibits structural similarity to that of the UDP–glucose glycoprotein glucosyltransferase. Comparison with UDP–glucose glycoprotein glucosyltransferase also revealed a putative donor substrate-binding site, and the functional requirements of critical residues in human GNPTAB were validated using GNPTAB-knockout cells. Finally, we show that DmGNPTAB forms a homodimer that is evolutionarily conserved and that perturbing the dimer interface undermines the maturation and activity of human GNPTAB. These results provide important insights into GlcNAc-1-phosphotransferase function and related diseases. 相似文献
2.
Properties of Solubilized UDP-GlcNAc: Dolichyl Phosphate-GlcNAc-1-P-Transferase from Soybean Cultured Cells 总被引:1,自引:1,他引:0
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The GlcNAc-1-P-transferase was solubilized from microsomal preparations of soybean cultured cells by treatment with 1% Triton X-100. The solubilized enzyme catalyzed the formation of dolichyl pyrophosphoryl-GlcNAc when incubated with UDP-GlcNAc and dolichyl phosphate. The GlcNAc-1-P-transferase activity was stimulated by the addition of phosphatidylglycerol and phosphatidylinositol, but was inhibited by phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The Km value for dolichyl-phosphate was 6.2 micromolar and that determined for UDP-GlcNAc was 0.42 micromolar. The pH optimum for the GlcNAc-1-P reaction was between 7.2 and 7.6; maximum activity occurred at about 10 millimolar Mg2+. The addition of unlabeled GDP-mannose or UDP-glucose considerably inhibited enzyme activity which could be restored to nearly the original value by addition of more dolichyl phosphate to the incubation mixture. On the other hand, the addition of unlabeled ADP-glucose and GDP-glucose enhanced the enzyme activity. This stimulation by these sugar nucleotides was found to be due to the protection of the substrate UDP-[3H]-GlcNAc from pyrophosphatase degradation. The GlcNAc-1-P-transferase reaction was very sensitive to tunicamycin and 50% inhibition required less than 1 microgram of antibiotic per milliliter. Amphomycin, showdomycin, and diumycin also inhibited this reaction but at higher concentrations. 相似文献
3.
R. Tauber R. Nuck W. Gerok R. Büchsel E. Köttgen W. Lohle Ch. Karasiewicz W. Reutter 《Glycoconjugate journal》1992,9(3):132-140
The effect of vitamin A deficiency onN-linked oligosaccharides of membrane glycoproteins was studied in rat liver in order to evaluate the suggested role of retinol in proteinN-glycosylation. First, oligosaccharides of newly synthesized glycoproteins from rough endoplasmic reticulum of vitamin A deficient liver were compared with that of pair-fed controls. Oligosaccharides were metabolically labelled withd-[2-3H]mannose, released from the glycoproteins with endoglycosidase H, purified by reversed phase HPLC and ion exchange chromatography, and were reduced with sodium borohydride. HPLC fractionation of the oligosaccharide alditols showed that the glycoproteins carried mainly four oligosaccharide species, Glc1Man9GlcNAc2, Man9GlcNAc2, Man8GlcNAc2 and Man7GlcNAc2, in identical relative amounts in the vitamin A deficient and the control tissue. In particular, no increase in the proportion of short chain oligosaccharides was noted in vitamin A deficient liver. Second, the number ofN-linked oligosaccharides was estimated in dipeptidylpeptidase IV (DPP IV), a major glycoprotein constituent of the hepatic plasma membrane, comparing the newly synthesized glycoprotein from rough endoplasmic reticulum and the mature form of DPP IV from the plasma membrane. No evidence was obtained that retinol deficiency caused incomplete glycosylation of this membrane glycoprotein. From these data, the suggested role of retinol as a cofactor involved in the synthesis ofN-linked oligosaccharides of glycoproteins must be questioned.Abbreviations DolP
Dolichyl phosphate
- DolPP
dolichyl pyrophosphoryl
- RetPMan
retinyl phosphate mannose
- DPP IV
dipeptidyl peptidase IV (EC 3.4.14.5)
- endo H
endo--N-acetylglucosaminidase H (EC 3.2.1.96)
- endo F
endo--N-acetylglucosaminidase F (EC 3.2.1.96)
- SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis 相似文献
4.
Freshly prepared protoplasts of Saccharomyces cerevisiae X 2180 incorporate [3H]mannose and [14C]glucose for about 30 min into glycolipids and mannoproteins. Among the radioactive glycolipids formed dolichyl phosphate mannose, dolichyl phosphate glucose and dolichyl pyrophosphate oligosaccharides have been identified. The oligosaccharides released by weak acid from the dolichyl pyrophosphate were treated with endo-N-acetylglucosaminidase H and separated by gel filtration on Bio-Gel P-4. The largest oligosaccharide obtained corresponded exactly in size to Glc3Man9GlcNAc1 the compound formed also in animal tissues. Other oligosaccharides released from dolichyl pyrophosphate in addition to the glucose containing ones were mainly Man9GlcNAc1 and Man8GlcNAc1. No mannosyl oligosaccharide corresponding in size to the total inner core region found in native mannoproteins could be detected in a lipid-bound form.The radioactive dolichyl pyrophosphate oligosaccharides were formed transiently; after 40 min only about 40% of the maximal radioactivity was observed in this fraction. In the presence of cycloheximide this decrease did not take place.It is concluded that the dolichol pathway of N-glycosylation of glycoproteins in yeast cells is very similar, if not identical, to the reaction sequence worked out for animal cells.Dedicated to Professor Dr. Otto Kandler on his 60th birthday 相似文献
5.
《Biochimica et Biophysica Acta (BBA)/General Subjects》1986,884(3):497-501
The availability of dolichyl phosphate is a major factor in the rate of formation of N-linked glycoproteins in mammalian cells. Recent studies in our laboratory suggested that glycoproteins required for seed germination and early plant development are formed via the dolichyl phosphate pathway. Soybean microsomes contain dolichol kinase and dolichyl phosphate phosphatase, enzymes that regulate dolichyl phosphate levels by interconversion of dolichyl phosphate and dolichol. In the present study, soybean microsomes were fractionated into rough and smooth endoplasmic reticulum and Golgi, and the activities of dolichol kinase and dolichyl phosphate phosphatase were measured in each. Submicrosomal fractions were obtained using a procedure developed for rat liver, and were characterized by marker enzymes, RNA content and electron microscopy. The site of N-glycosylation, the rough endoplasmic reticulum, contained high levels of both dolichol kinase and dolichyl phosphate phosphatase. This makes possible a mechanism whereby glycoprotein formation during seed germination is regulated by availability of dolichyl phosphate. 相似文献
6.
Marielle Boonen Peter Vogel Kenneth A. Platt Nancy Dahms Stuart Kornfeld 《Molecular biology of the cell》2009,20(20):4381-4389
The mannose 6-phosphate (Man-6-P) lysosomal targeting signal on acid hydrolases is synthesized by the sequential action of uridine 5′-diphosphate-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) and GlcNAc-1-phosphodiester α-N-acetylglucosaminidase (“uncovering enzyme” or UCE). Mutations in the two genes that encode GlcNAc-1-phosphotransferase give rise to lysosomal storage diseases (mucolipidosis type II and III), whereas no pathological conditions have been associated with the loss of UCE activity. To analyze the consequences of UCE deficiency, the UCE gene was inactivated via insertional mutagenesis in mice. The UCE −/− mice were viable, grew normally and lacked detectable histologic abnormalities. However, the plasma levels of six acid hydrolases were elevated 1.6- to 5.4-fold over wild-type levels. These values underestimate the degree of hydrolase hypersecretion as these enzymes were rapidly cleared from the plasma by the mannose receptor. The secreted hydrolases contained GlcNAc-P-Man diesters, exhibited a decreased affinity for the cation-independent mannose 6-phosphate receptor and failed to bind to the cation-dependent mannose 6-phosphate receptor. These data demonstrate that UCE accounts for all the uncovering activity in the Golgi. We propose that in the absence of UCE, the weak binding of the acid hydrolases to the cation-independent mannose 6-phosphate receptor allows sufficient sorting to lysosomes to prevent the tissue abnormalities seen with GlcNAc-1-phosphotranferase deficiency. 相似文献
7.
Sandra Pohl Stephan Tiede Katrin Marschner Marisa Encarna??o Monica Castrichini Katrin Kollmann Nicole Muschol Kurt Ullrich Sven Müller-Loennies Thomas Braulke 《The Journal of biological chemistry》2010,285(31):23936-23944
GlcNAc-1-phosphotransferase is a Golgi-resident 540-kDa complex of three subunits, α2β2γ2, that catalyze the first step in the formation of the mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes. Anti-M6P antibody analysis shows that human primary macrophages fail to generate M6P residues. Here we have explored the sorting and intracellular targeting of cathepsin D as a model, and the expression of the GlcNAc-1-phosphotransferase complex in macrophages. Newly synthesized cathepsin D is transported to lysosomes in an M6P-independent manner in association with membranes whereas the majority is secreted. Realtime PCR analysis revealed a 3–10-fold higher GlcNAc-1-phosphotransferase subunit mRNA levels in macrophages than in fibroblasts or HeLa cells. At the protein level, the γ-subunit but not the β-subunit was found to be proteolytically cleaved into three fragments which form irregular 97-kDa disulfide-linked oligomers in macrophages. Size exclusion chromatography showed that the γ-subunit fragments lost the capability to assemble with other GlcNAc-1-phosphotransferase subunits to higher molecular complexes. These findings demonstrate that proteolytic processing of the γ-subunit represents a novel mechanism to regulate GlcNAc-1-phosphotransferase activity and the subsequent sorting of lysosomal enzymes. 相似文献
8.
Primary structural requirements both for N- and O-glycosylation have been studied using a series of synthetic peptides and a membrane fraction from Saccharomyces cerevisiae. N-Glycosylation: the tripeptide sequence Asn-Xaa-Thr/Ser was found to be necessary for the transfer of saccharide units from oligosaccharide-lipid to asparagine. Substitution of asparagine by aspartic acid or glutamine, or replacement of threonine by valine in the hexapeptide Tyr--Leu--Ser-Val prevents its glycosylation. Also, a proline residue in the position of Xaa makes the peptide unable to function as an acceptor. Transfer onto asparagine occurs only efficiently if both the α-amino group of asparagine and the α-carboxyl moiety of the hydroxy amino acid are blocked. Yield of glycosylation improves with increasing peptide chain length. With regard to the glycosyl donor dolichyl diphosphate-bound GlcNAc2Man9Glc3 is the preferred substrate. Non-glucosylated glycolipid Dol-PP-GlcNAc2Man9 is a poor donor, whereas smaller precursors Dol-PP-GlcNAc2 and Dol-PP-GlcNAc2Man1 allow reasonable transfer. O-Glycosylation: no marker sequence can be derived for the formation of an O-glycosidic linkage via Dol-P-Man. Introduction of a proline residue in vicinity to the hydroxy amino acid leads to a significant improvement of glycosyl transfer. It is postulated that accessibility of potential O-glycosylation sites rather than a specific sequence may be a prerequisite for O-glycosylation. 相似文献
9.
Shams-Eldin H Chaban B Niehus S Schwarz RT Jarrell KF 《Journal of bacteriology》2008,190(6):2217-2220
The Mv1751 gene product is thought to catalyze the first step in the N-glycosylation pathway in Methanococcus voltae. Here, we show that a conditional lethal mutation in the alg7 gene (N-acetylglucosamine-1-phosphate transferase) in Saccharomyces cerevisiae was successfully complemented with Mv1751, highlighting a rare case of cross-domain complementation. 相似文献
10.
A. Phongdara A. Merckelbach P. Keup G. Gellissen C. P. Hollenberg 《Applied microbiology and biotechnology》1998,50(1):77-84
A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA
fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with
a calculated M
r of 49400. The␣encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide
sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved
in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression.
Received: 15 January 1998 / Received revision: 2 March 1998 / Accepted: 4 March 1998 相似文献
11.
M.N. Heneghan L. McLoughlin P.G. Murray M.G. Tuohy 《Enzyme and microbial technology》2007,41(6-7):677-682
The aguA gene encoding α-glucuronidase was isolated from the thermophilic fungus Talaromyces emersonii by degenerate PCR. AguA has no introns and consists of an open reading frame of 2511 bp, encoding a putative protein of 837 amino acids. The N-terminus of the protein contains a putative signal peptide of 17 amino acids yielding a mature protein of 820 amino acids with a predicted molecular mass of 91.6 kDa. Twenty putative N-glycosylation sites and four O-glycosylation were identified. The T. emersonii α-glucuronidase falls into glycosyl hydrolase family 67, showing approximately 63% identity to similar enzymes from other fungi. Analysis of the aguA promoter revealed several possible regulatory motifs including two XlnR and a CreA binding site. Enzyme activity was optimal at 50 °C and pH 5. Enzyme production was investigated on a range of carbon sources and showed induction on beechwood, oat spelt and birchwood xylan, and repression by glucose or glucuronic acid. 相似文献
12.
This study concerns inter- and intraspecific differences between yeasts at assimilation of different nitrogen sources. Alterations in the content of free amino acids in cells and media as well as in the related enzyme activities during growth were studied. The hydroxylamine (HA)-tolerant Endomycopsis lipolytica was examined and compared with the nitrate-reducing Cryptococcus albidus, and Saccharomyces cerevisiae, requiring fully reduced nitrogen for growth. Special attention was paid to alanine, aspartic acid, and glutamic acid, the amino acids closely related to the Krebs cycle keto acids. The amino acids were analyzed as their n-propyl N-acetyl esters by gas-liquid chromatography (GLC).The composition of the amino acid pool was similar for the three yeasts. Glutamic acid was predominant; in early log-phase cells of E. lipolytica contents of 200–234 mol·g-1 dry weight were found. A positive correlation between the specific growth rate and the size of the amino acid pool was observed.The assimilation of ammonia was mediated by glutamate dehydrogenase (GDH). The NADP-GDH was the dominating enzyme in all three yeasts showing the highest specific activity in Cr. albidus grown on nitrate (6980 nmol· (min-1)·(mg protein-1). Glutamine synthetase (GS) displayed a high specific activity in S. cerevisiae, which also had a high amount of glutamine. The assimilation of HA did not differ greatly from the assimilation of ammonium in E. lipolytica. The existing differences could rather be explained as provoked by the concentration of available nitrogen. 相似文献
13.
《The International journal of biochemistry》1986,18(2):195-198
- 1.1. A maximum rate of dolichyl phosphate [14C]glucose synthesis from 55-day embryos was achieved at 16nM concentration of exogenous dolichyl phosphate and exceeded about 3 times that without addition of dolichyl phosphate.
- 2.2. The highest values of [14C]glucose incorporation from UDP-[14C]glucose into dolichyl phosphate [14C]glucose, dolichyl diphosphate [14C]Glc-oligosaccharides and proteins were reached at 5 min time point of incubation of liver microsomes both from embryos and sows.
- 3.3. The radioactive incorporation into proteins was about 7-fold higher in liver microsomes from sows compared to that from embryos, probably due to the greater content of acceptor proteins in microsomes from sows.
- 4.4. The enzymatic transfer of Glc3-oligosaccharide from a lipid carrier to endogenous protein acceptor in microsomes from pig embryonic and adult livers was considerably faster than the removal of glucose residues during the initial stages of processing of protein-bound oligosaccharides.
- 5.5. One labelled compound was discovered in the Chcl3-Ch3Oh-H2O (1:1:0.3, by vol) extract after incubation of liver microsomes from embryos and sows with UDP-[14C]glucose. On the basis of its mobility on the chromatogram it appears to be GlcNAc2Man9Glc3.
14.
Kim JH Campbell BC Yu J Mahoney N Chan KL Molyneux RJ Bhatnagar D Cleveland TE 《Applied microbiology and biotechnology》2005,67(6):807-815
Saccharomyces cerevisiae served as a model fungal system to examine functional genomics of oxidative stress responses and reactions to test antioxidant compounds. Twenty-two strains of S. cerevisiae, including a broad spectrum of singular gene deletion mutants, were exposed to hydrogen peroxide (H2O2) to examine phenotypic response to oxidative stress. Responses of particular mutants treated with gallic, tannic or caffeic acids, or methyl gallate, during H2O2 exposure, indicated that these compounds alleviated oxidative stress. These compounds are also potent inhibitors of aflatoxin biosynthesis in Aspergillus flavus. To gain further insights into a potential link between oxidative stress and aflatoxin biosynthesis, 43 orthologs of S. cerevisiae genes involved in gene regulation, signal transduction (e.g., SHO1, HOG1, etc.) and antioxidation (e.g., CTT1, CTA1, etc.) were identified in an A. flavus expressed sequence tag library. A successful exemplary functional complementation of an antioxidative stress gene from A. flavus, mitochondrial superoxide dismutase (sodA), in a sod2 yeast mutant further supported the potential of S. cerevisiae deletion mutants to serve as a model system to study A. flavus. Use of this system to further examine functional genomics of oxidative stress in aflatoxigenesis and reduction of aflatoxin biosynthesis by antioxidants is discussed. 相似文献
15.
Summary We report here the isolation of temperature-sensitive mutants of the yeast Saccharomyces cerevisiae which exhibit cdc phenotypes. The recessive mutations defined four complementation groups, named ore1, ore2, ore3 and ore4. At the non-permissive temperature, strains bearing these mutations arrested in the G1 phase of the cell cycle. The wild-type allele of the gene altered in ore2 mutants was cloned. The nucleotide sequence of a fragment which can complement the mutation showed the presence of an open reading frame capable of encoding a protein with 286 amino acid residues. The deduced amino acid sequence showed 25% identity with that of the Escherichia coli 1-pyrroline-5-carboxylate reductase, an enzyme of the pathway for the biosynthesis of proline. The ore2 mutants, correspondingly, were found to be capable of growing at the non-permissive temperature on a synthetic medium supplemented with proline. In addition, the chromosomal location of the gene and its restriction map were compatible with those previously reported for the PRO3 gene which encodes the S. cerevisiae 1-pyrroline-5-carboxylate reductase. 相似文献
16.
Mucolipidoses (ML) II and III alpha/beta are lysosomal storage diseases caused by pathogenic mutations in GNPTAB encoding the α⁄β-subunit precursor of GlcNAc-1-phosphotransferase. To determine genotype-phenotype correlation and functional analysis of mutant GlcNAc-1-phosphotransferase, 13 Brazilian patients clinically and biochemical diagnosed for MLII or III alpha/beta were studied. By sequencing of genomic GNPTAB of the MLII and MLIII alpha/beta patients we identified six novel mutations: p.D76G, p.S385L, p.Q278Kfs*3, p.H588Qfs*27, p.N642Lfs*10 and p.Y1111*. Expression analysis by western blotting and immunofluorescence microscopy revealed that the mutant α⁄β-subunit precursor p.D76G is retained in the endoplasmic reticulum whereas the mutant p.S385L is correctly transported to the cis-Golgi apparatus and proteolytically processed. Both mutations lead to complete loss of GlcNAc-1-phosphotransferase activity, consistent with the severe clinical MLII phenotype of the patients. Our study expands the genotypic spectrum of MLII and provides novel insights into structural requirements to ensure GlcNAc-1-phosphotransferase activity. 相似文献
17.
Mucolipidosis II is caused by mutations in GNPTA encoding the alpha/beta GlcNAc-1-phosphotransferase
Tiede S Storch S Lübke T Henrissat B Bargal R Raas-Rothschild A Braulke T 《Nature medicine》2005,11(10):1109-1112
Mucolipidosis II (ML II) is a fatal lysosomal storage disorder resulting from defects in the multimeric GlcNAc-1-phosphotransferase responsible for the initial step in the generation of the mannose 6-phosphate (M6P) recognition marker. M6P residues on oligosaccharides of newly synthesized lysosomal enzymes are essential for efficient receptor-mediated transport to lysosomes. We used the recombinant GlcNAc-1-phosphotransferase gamma subunit as an affinity matrix to purify an unknown protein identified as the product of GNPTA (encoding GNPTA, previously known as MGC4170). The cDNA encodes a protein of 1,256 amino acids with two putative transmembrane domains and a complex preserved modular structure comprising at least six domains. The N-terminal domain of GNPTA, interrupted by a long insertion, shows similarities to bacterial capsule biosynthesis proteins. We identified seven mutations in GNPTA that lead to premature translational termination in six individuals with ML II. Retroviral transduction of fibroblasts from an individual with ML II resulted in the expression and localization of GNPTA in the Golgi apparatus, accompanied by the correction of hypersecretion of lysosomal enzymes. Our results provide evidence that GNPTA encodes a subunit of GlcNAc-1-phosphotransferase defective in individuals with ML II. 相似文献
18.
Nam YD Chang HW Kim KH Roh SW Kim MS Jung MJ Lee SW Kim JY Yoon JH Bae JW 《Journal of microbiology (Seoul, Korea)》2008,46(5):491-501
The bacterial, archaeal, and eukaryal diversity in fecal samples from ten Koreans were analyzed and compared by using the
PCR-fingerprinting method, denaturing gradient gel electrophoresis (DGGE). The bacteria all belonged to the Firmicutes and Bacteroidetes phyla, which were known to be the dominant bacterial species in the human intestine. Most of the archaeal sequences belonged
to the methane-producing archaea but several halophilic archarea-related sequences were also detected unexpectedly. While
a small number of eukaryal sequences were also detected upon DGGE analysis, these sequences were related to fungi and stramenopiles
(Blastocystis hominis). With regard to the bacterial and archaeal DGGE analysis, all ten samples had one and two prominent bands, respectively,
but many individual-specific bands were also observed. However, only five of the ten samples had small eukaryal DGGE bands
and none of these bands was observed in all five samples. Unweighted pair group method and arithmetic averages clustering
algorithm (UPGMA) clustering analysis revealed that the archaeal and bacterial communities in the ten samples had relatively
higher relatedness (the average Dice coefficient values were 68.9 and 59.2% for archaea and bacteria, respectively) but the
eukaryal community showed low relatedness (39.6%). 相似文献
19.
20.
Yi Qian Eline van Meel Heather Flanagan-Steet Alex Yox Richard Steet Stuart Kornfeld 《The Journal of biological chemistry》2015,290(5):3045-3056
UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase tags newly synthesized lysosomal enzymes with mannose 6-phosphate recognition markers, which are required for their targeting to the endolysosomal system. GNPTAB encodes the α and β subunits of GlcNAc-1-phosphotransferase, and mutations in this gene cause the lysosomal storage disorders mucolipidosis II and III αβ. Prior investigation of missense mutations in GNPTAB uncovered amino acids in the N-terminal region and within the DMAP domain involved in Golgi retention of GlcNAc-1-phosphotransferase and its ability to specifically recognize lysosomal hydrolases, respectively. Here, we undertook a comprehensive analysis of the remaining missense mutations in GNPTAB reported in mucolipidosis II and III αβ patients using cell- and zebrafish-based approaches. We show that the Stealth domain harbors the catalytic site, as some mutations in these regions greatly impaired the activity of the enzyme without affecting its Golgi localization and proteolytic processing. We also demonstrate a role for the Notch repeat 1 in lysosomal hydrolase recognition, as missense mutations in conserved cysteine residues in this domain do not affect the catalytic activity but impair mannose phosphorylation of certain lysosomal hydrolases. Rescue experiments using mRNA bearing Notch repeat 1 mutations in GNPTAB-deficient zebrafish revealed selective effects on hydrolase recognition that differ from the DMAP mutation. Finally, the mutant R587P, located in the spacer between Notch 2 and DMAP, was partially rescued by overexpression of the γ subunit, suggesting a role for this region in γ subunit binding. These studies provide new insight into the functions of the different domains of the α and β subunits. 相似文献