Proteomics Analysis of Serum from Mutant Mice Reveals Lysosomal Proteins Selectively Transported by Each of the Two Mannose 6-Phosphate Receptors

Qian, Meiqian; Sleat, David E.; Zheng, Haiyan; Moore, Dirk F.; Lobel, Peter

doi:10.1074/mcp.m700217-mcp200

Cited by 79 publications

(70 citation statements)

References 59 publications

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“…Each of the three genotypes were processed in parallel essentially as described previously (23). Tissue was diluted into 9 volumes (w/v) of homogenization buffer (20 mM sodium phosphate, pH 6.8, 150 mM NaCl, 2.5 mM EDTA, 5 mM ␤-glycerophosphate, 1 g/ml leupeptin, 1 g/ml pepstatin A) containing 1 mM Pefabloc and 1% Triton X-100 and disrupted using a Brinkmann Polytron.…”

Section: Materials-udp-[6-mentioning

confidence: 99%

Functions of the α, β, and γ Subunits of UDP-GlcNAc:Lysosomal Enzyme N-Acetylglucosamine-1-phosphotransferase

Qian

Lee

et al. 2010

Journal of Biological Chemistry

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UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an ␣ 2 ␤ 2 ␥ 2 hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Using a multifaceted approach, including analysis of acid hydrolase phosphorylation in mice and fibroblasts lacking the ␥ subunit along with kinetic studies of recombinant ␣ 2 ␤ 2 ␥ 2 and ␣ 2 ␤ 2 forms of the transferase, we have explored the function of the ␣/␤ and ␥ subunits. The findings demonstrate that the ␣/␤ subunits recognize the protein determinant of acid hydrolases in addition to mediating the catalytic function of the transferase. In mouse brain, the ␣/␤ subunits phosphorylate about one-third of the acid hydrolases at close to wild-type levels but require the ␥ subunit for optimal phosphorylation of the rest of the acid hydrolases. In addition to enhancing the activity of the ␣/␤ subunits toward a subset of the acid hydrolases, the ␥ subunit facilitates the addition of the second GlcNAc-P to high mannose oligosaccharides of these substrates. We postulate that the mannose 6-phosphate receptor homology domain of the ␥ subunit binds and presents the high mannose glycans of the acceptor to the ␣/␤ catalytic site in a favorable manner.In higher eukaryotes, newly synthesized acid hydrolases acquire mannose 6-phosphate (Man-6-P) 3 residues on their N-linked glycans as they traverse the Golgi (1). These residues serve as high affinity ligands for binding to Man-6-P receptors in the trans-Golgi network. The hydrolase-receptor complexes are then packaged into transport carriers for delivery to endosomes and lysosomes. The Man-6-P recognition marker is synthesized in two steps. First, UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) binds to a conformation-dependent protein determinant on the acid hydrolase and transfers GlcNAc-1-P from UDP-GlcNAc to one or two of the mannose residues of the N-linked high mannose oligosaccharide. Second, N-acetylglucosamine 1-phosphodiester ␣-N-acetyglucosaminidase ("uncovering enzyme") excises the N-acetylglucosamine to generate the Man-6-P monoester.GlcNAc-1-phosphotransferase is a heterohexamer composed of three subunits (␣ 2 ␤ 2 ␥ 2 ) (2). The ␣ and ␤ subunits are encoded by a single gene GNPTAB, and the ␥ subunit is encoded by a separate gene GNPTG (3-5). Although it has been established that the ␣/␤ subunits contain the catalytic activity of the enzyme, the possible participation of these subunits in the recognition of the common protein determinant of the acid hydrolases has not been explored. Furthermore, the role(s) of the ␥ subunit is poorly understood. The initial insight into the function of the subunits came from studies of patients with the autosomal recessive lysosomal storage disorders termed mucolipidosis II (I-cell disease) and mucolipidosis III (pseudo-Hurler polydystrophy), the latter being the less severe of the two (6). These disorders arise from mutations in the genes encoding GlcNAc-1-phospho...

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Section: Materials-udp-[6-mentioning

confidence: 99%

Functions of the α, β, and γ Subunits of UDP-GlcNAc:Lysosomal Enzyme N-Acetylglucosamine-1-phosphotransferase

Qian

Lee

et al. 2010

Journal of Biological Chemistry

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“…This sequential process of N-glycan modification may relate to the existence of two different MPRs that appear to have different specificity and modes of interactions (15). The 300-kDa CI-MPR contains 15 homologous, contiguous MRH (mannose 6-phosphate receptor homology) domains, three of which, domains 3, 5, and 9, contain a Man-6-P binding site (16 -18).…”

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confidence: 99%

Glycan Microarray Analysis of P-type Lectins Reveals Distinct Phosphomannose Glycan Recognition

Song

Lasanajak

Olson

et al. 2009

Journal of Biological Chemistry

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The specificity of the cation-independent and -dependent mannose 6-phosphate receptors (CI-MPR and CD-MPR) for high mannose-type N-glycans of defined structure containing zero, one, or two Man-P-GlcNAc phosphodiester or Man-6-P phosphomonoester residues was determined by analysis on a phosphorylated glycan microarray. Amine-activated glycans were covalently printed on N-hydroxysuccinimide-activated glass slides and interrogated with different concentrations of recombinant CD-MPR or soluble CI-MPR. Neither receptor bound to non-phosphorylated glycans. The CD-MPR bound weakly or undetectably to the phosphodiester derivatives, but strongly to the phosphomonoester-containing glycans with the exception of a single Man7GlcNAc2-R isomer that contained a single Man-6-P residue. By contrast, the CI-MPR bound with high affinity to glycans containing either phospho-mono-or -diesters although, like the CD-MPR, it differentially recognized isomers of phosphorylated Man7GlcNAc2-R. This differential recognition of phosphorylated glycans by the CI-and CD-MPRs has implications for understanding the biosynthesis and targeting of lysosomal hydrolases.

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“…This protein is present in plasma and cerebrospinal fluid and may be involved in the pathogenesis of Alzheimer disease as it appears to prevent proteolysis of amyloid fibrils (48,49). It was previously found in preparations of Man6-P glycoproteins from human and mouse plasma (50,51) as well as a wide range of rat tissues (23) although the presence of Man6-P has not been directly demonstrated. Interestingly, there is evidence that APCS can bind carbohydrates and proteins containing Man6-P (52) which raises the possibility that this protein may not be a Man6-P glycoprotein per se but may be transported to the lysosome by MPR-mediated endocytosis while in association with other proteins containing Man6-P.…”

Section: Discussionmentioning

confidence: 99%

Classification of Subcellular Location by Comparative Proteomic Analysis of Native and Density-shifted Lysosomes

Valle

Sleat

Zheng

et al. 2011

Molecular & Cellular Proteomics

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One approach to the functional characterization of the lysosome lies in the use of proteomic methods to identify proteins in subcellular fractions enriched for this organelle. However, distinguishing between true lysosomal residents and proteins from other cofractionating organelles is challenging. To this end, we implemented a quantitative mass spectrometry approach based on the selective decrease in the buoyant density of liver lysosomes that occurs when animals are treated with Triton-WR1339. Liver lysosome-enriched preparations from control and treated rats were fractionated by isopycnic sucrose density gradient centrifugation. Tryptic peptides derived from gradient fractions were reacted with isobaric tag for relative and absolute quantitation eight-plex labeling reagents and analyzed by two-dimensional liquid chromatography matrix-assisted laser desorption ionization time-of-flight MS. Reporter ion intensities were used to generate relative protein distribution profiles across both types of gradients. A distribution index was calculated for each identified protein and used to determine a probability of lysosomal residence by quadratic discriminant analysis. This analysis suggests that several proteins assigned to the lysosome in other proteomics studies are not true lysosomal residents. Conversely, results support lysosomal residency for other proteins that are either not or only tentatively assigned to this location. The density shift for two proteins, Cu/Zn superoxide dismutase and ATP-binding cassette subfamily B (MDR/TAP) member 6, was corroborated by quantitative Western blotting. Additional balance sheet analyses on differential centrifugation fractions revealed that Cu/Zn superoxide dismutase is predominantly cytosolic with a secondary lysosomal localization whereas ATP-binding cassette subfamily B (MDR/TAP) member 6 is predominantly lysosomal. These results establish a quantitative mass spectrometric/subcellular fractionation approach for identification of lysosomal proteins and underscore

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Proteomics Analysis of Serum from Mutant Mice Reveals Lysosomal Proteins Selectively Transported by Each of the Two Mannose 6-Phosphate Receptors

Cited by 79 publications

References 59 publications

Functions of the α, β, and γ Subunits of UDP-GlcNAc:Lysosomal Enzyme N-Acetylglucosamine-1-phosphotransferase

Functions of the α, β, and γ Subunits of UDP-GlcNAc:Lysosomal Enzyme N-Acetylglucosamine-1-phosphotransferase

Glycan Microarray Analysis of P-type Lectins Reveals Distinct Phosphomannose Glycan Recognition

Classification of Subcellular Location by Comparative Proteomic Analysis of Native and Density-shifted Lysosomes

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