A Lifetime of Adventures in Glycobiology.

My initial research experience involved studying how bacteria synthesize nucleotide sugars, the donors for the formation of cell wall polysaccharides. During this time, I became aware that mammalian cells also have a surface coat of sugars and was intrigued as to whether these sugars might be arranged in specific sequences that function as information molecules in biologic processes. Thus began a long journey that has taken me from glycan structural analysis and determination of plant lectin-binding preferences to the biosynthesis of Asn-linked oligosaccharides and the mannose 6-phosphate (Man-6-P) lysosomal enzyme targeting pathway. The Man-6-P system represents an early example of a glycan serving as an information molecule in a fundamental cellular function. The remarkable advances in the field of glycobiology since I entered have uncovered scores of additional examples of oligosaccharide-lectin interactions mediating critical biologic processes. It has been a rewarding experience to participate in the efforts that have established a central role for glycans in biology.

Structures of the mannose-6-phosphate pathway enzyme, GlcNAc-1-phosphotransferase.

The mannose-6-phosphate (M6P) pathway is responsible for the transport of hydrolytic enzymes to lysosomes. N-acetylglucosamine-1-phosphotransferase (GNPT) catalyzes the first step of tagging these hydrolases with M6P, which when recognized by receptors in the Golgi diverts them to lysosomes. Genetic defects in the GNPT subunits, GNPTAB and GNPTG, cause the lysosomal storage diseases mucolipidosis types II and III. To better understand its function, we determined partial three-dimensional structures of the GNPT complex. The catalytic domain contains a deep cavity for binding of uridine diphosphate-N-acetylglucosamine, and the surrounding residues point to a one-step transfer mechanism. An isolated structure of the gamma subunit of GNPT reveals that it can bind to mannose-containing glycans in different configurations, suggesting that it may play a role in directing glycans into the active site. These findings may facilitate the development of therapies for lysosomal storage diseases.

Response to Comments on "Increasing Enzyme Mannose-6-Phosphate Levels but Not Miglustat Coadministration Enhances the Efficacy of Enzyme Replacement Therapy in Pompe Mice".

We thank Dr. Weimer and her colleagues for their comments related to our recent work (Anding et al., 2023) and are grateful for the opportunity to further discuss the importance of efficient lysosomal targeting of enzyme-replacement therapies (ERT) for the treatment of Pompe disease. Patients with Pompe disease have mutations in the gene that encodes for acid α glucosidase (GAA), a lysosomal enzyme necessary for the breakdown of glycogen. The first-generation ERT, alglucosidase alfa, provides a lifesaving therapy for the severe form of the disease (infantile onset Pompe disease) and improves or stabilizes respiratory and motor function in patients with less severe disease (late onset Pompe disease). Despite these gains, significant unmet need remains, particularly in patients who display respiratory and motor decline following years of treatment. Poor tissue uptake and lysosomal targeting via inefficient binding of the cation-independent mannose-6-phosphate (M6P) receptor (CIMPR) in skeletal muscle contributed to this suboptimal treatment response, prompting the development of new ERTs with increased levels of M6P.

Liposome-encapsulated mannose-1-phosphate therapy improves global N-glycosylation in different congenital disorders of glycosylation.

Phosphomannomutase 2 (PMM2) converts mannose-6-phospahate to mannose-1-phosphate; the substrate for GDP-mannose, a building block of the glycosylation biosynthetic pathway. Pathogenic variants in the PMM2 gene have been shown to be associated with protein hypoglycosylation causing PMM2-congenital disorder of glycosylation (PMM2-CDG). While mannose supplementation improves glycosylation in vitro, but not in vivo, we hypothesized that liposomal delivery of mannose-1-phosphate could increase the stability and delivery of the activated sugar to enter the targeted compartments of cells. Thus, we studied the effect of liposome-encapsulated mannose-1-P (GLM101) on global protein glycosylation and on the cellular proteome in skin fibroblasts from individuals with PMM2-CDG, as well as in individuals with two N-glycosylation defects early in the pathway, namely ALG2-CDG and ALG11-CDG. We leveraged multiplexed proteomics and N-glycoproteomics in fibroblasts derived from different individuals with various pathogenic variants in PMM2, ALG2 and ALG11 genes. Proteomics data revealed a moderate but significant change in the abundance of some of the proteins in all CDG fibroblasts upon GLM101 treatment. On the other hand, N-glycoproteomics revealed the GLM101 treatment enhanced the expression levels of several high-mannose and complex/hybrid glycopeptides from numerous cellular proteins in individuals with defects in PMM2 and ALG2 genes. Both PMM2-CDG and ALG2-CDG exhibited several-fold increase in glycopeptides bearing Man6 and higher glycans and a decrease in Man5 and smaller glycan moieties, suggesting that GLM101 helps in the formation of mature glycoforms. These changes in protein glycosylation were observed in all individuals irrespective of their genetic variants. ALG11-CDG fibroblasts also showed increase in high mannose glycopeptides upon treatment; however, the improvement was not as dramatic as the other two CDG. Overall, our findings suggest that treatment with GLM101 overcomes the genetic block in the glycosylation pathway and can be used as a potential therapy for CDG with enzymatic defects in early steps in protein N-glycosylation.

Mannose impairs tumour growth and enhances chemotherapy.

It is now well established that tumours undergo changes in cellular metabolism1. As this can reveal tumour cell vulnerabilities and because many tumours exhibit enhanced glucose uptake2, we have been interested in how tumour cells respond to different forms of sugar. Here we report that the monosaccharide mannose causes growth retardation in several tumour types in vitro, and enhances cell death in response to major forms of chemotherapy. We then show that these effects also occur in vivo in mice following the oral administration of mannose, without significantly affecting the weight and health of the animals. Mechanistically, mannose is taken up by the same transporter(s) as glucose3 but accumulates as mannose-6-phosphate in cells, and this impairs the further metabolism of glucose in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway and glycan synthesis. As a result, the administration of mannose in combination with conventional chemotherapy affects levels of anti-apoptotic proteins of the Bcl-2 family, leading to sensitization to cell death. Finally we show that susceptibility to mannose is dependent on the levels of phosphomannose isomerase (PMI). Cells with low levels of PMI are sensitive to mannose, whereas cells with high levels are resistant, but can be made sensitive by RNA-interference-mediated depletion of the enzyme. In addition, we use tissue microarrays to show that PMI levels also vary greatly between different patients and different tumour types, indicating that PMI levels could be used as a biomarker to direct the successful administration of mannose. We consider that the administration of mannose could be a simple, safe and selective therapy in the treatment of cancer, and could be applicable to multiple tumour types.

Multivalent, Stabilized Mannose-6-Phosphates for the Targeted Delivery of Toll-Like Receptor Ligands and Peptide Antigens.

Mannose-6-phosphate (M6P) is recognized by the mannose-6-phosphate receptor and plays an important role in the transport of cargo to the endosomes, making it an attractive tool to improve endosomal trafficking of vaccines. We describe herein the assembly of peptide antigen conjugates carrying clusters of mannose-6-C-phosphonates (M6Po). The M6Po's are stable M6P mimics that are resistant to cleavage of the phosphate group by endogenous phosphatases. Two different strategies for the incorporation of the M6Po clusters in the conjugate have been developed: the first relies on a "post-assembly" click approach employing an M6Po bearing an alkyne functionality; the second hinges on an M6Po C-glycoside amino acid building block that can be used in solid-phase peptide synthesis. The generated conjugates were further equipped with a TLR7 ligand to stimulate dendritic cell (DC) maturation. While antigen presentation is hindered by the presence of the M6Po clusters, the incorporation of the M6Po clusters leads to increased activation of DCs, thus demonstrating their potential in improving vaccine adjuvanticity by intraendosomally active TLR ligands.

Targeted Analysis of Lysosomal Directed Proteins and Their Sites of Mannose-6-phosphate Modification.

Mannose-6-phosphate (M6P) is a distinctive post-translational modification critical for trafficking of lysosomal acid hydrolases into the lysosome. Improper trafficking into the lysosome, and/or lack of certain hydrolases, results in a toxic accumulation of their substrates within the lysosomes. To gain insight into the enzymes destined to the lysosome these glycoproteins can be distinctively enriched and studied using their unique M6P tag. Here we demonstrate, by adapting a protocol optimized for the enrichment of phosphopeptides using Fe3+-IMAC chromatography, that proteome-wide M6P glycopeptides can be selectively enriched and subsequently analyzed by mass spectrometry, taking advantage of exclusive phosphomannose oxonium fragment marker ions. As proof-of-concept of this protocol, applying it to HeLa cells, we identified hundreds of M6P-modified glycopeptides on 35 M6P-modified glycoproteins. We next targeted CHO cells, either wild-type or cells deficient in Acp2 and Acp5, which are acid phosphatases targeting M6P. In the KO CHO cells we observed a 20-fold increase of the abundance of the M6P-modification on endogenous CHO glycoproteins but also on the recombinantly over-expressed lysosomal human alpha-galactosidase. We conclude that our approach could thus be of general interest for characterization of M6P glycoproteomes as well as characterization of lysosomal enzymes used as treatment in enzyme replacement therapies targeting lysosomal storage diseases.

Insulin-Like Growth Factor 2 Receptor Expression Is Promoted by Human Herpesvirus 8-Encoded Interleukin-6 and Contributes to Viral Latency and Productive Replication.

Human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6) localizes largely to the endoplasmic reticulum (ER) and here associates functionally with both the gp130 signal transducer and the novel ER membrane protein vitamin K epoxide reductase complex subunit 1 variant-2 (VKORC1v2). The latter interaction contributes to the viability of latently infected primary effusion lymphoma (PEL) cells and to HHV-8 productive replication, in part via promotion of ER-associated degradation (ERAD) of nascent pro-cathepsin D (pCatD) and consequent suppression of lysosome-localized proapoptotic mature CatD. Here we report that VKORC1v2 associates with insulin-like growth factor 2 receptor (IGF2R), also known as cation-independent mannose-6-phosphate receptor, which is involved in trafficking of mannose-6-phosphate-conjugated glycoproteins to lysosomes. VKORC1v2 effected reduced IGF2R expression in a manner dependent on VKORC1v2-IGF2R interaction, while vIL-6, which could inhibit VKORC1v2-IGF2R interaction, effected increased expression of IGF2R. These effects were independent of changes in IGF2R mRNA levels, indicating likely posttranslational mechanisms. In kinetic analyses involving labeling of either newly synthesized or preexisting IGF2R, vIL-6 promoted accumulation of the former while having no detectable effect on the latter. Furthermore, vIL-6 led to decreased K48-linked ubiquitination of IGF2R and suppression of ERAD proteins effected increased IGF2R expression and loss of IGF2R regulation by vIL-6. Depletion-based experiments identified IGF2R as a promoter of PEL cell viability and virus yields from lytically reactivated cultures. Our findings identify ER-transiting nascent IGF2R as an interaction partner of VKORC1v2 and target of vIL-6 regulation and IGF2R as a positive contributor to HHV-8 biology, thereby extending understanding of the mechanisms of VKORC1v2-associated vIL-6 function.IMPORTANCE HHV-8 vIL-6 promotes productive replication in the context of reactivated lytic replication in primary effusion lymphoma (PEL) and endothelial cells and sustains latently infected PEL cell viability. Viral IL-6 is also considered to contribute significantly to HHV-8-associated pathogenesis, since vIL-6 can promote cell proliferation, cell survival, and angiogenesis that are characteristic of HHV-8-associated Kaposi's sarcoma, PEL and multicentric Castleman's disease (MCD), in addition to proinflammatory activities observed in MCD-like "Kaposi's sarcoma-associated herpesvirus-induced cytokine syndrome." We show in the present study that vIL-6 can promote productive replication and latent PEL cell viability through upregulation of the mannose-6-phosphate- and peptide hormone-interacting receptor IGF2R, which is a positive factor in HHV-8 biology via these activities. VKORC1v2-enhanced ER-associated degradation of IGF2R and vIL-6 promotion of IGF2R expression through prevention of its interaction with VKORC1v2 and consequent rescue from degradation represent newly recognized activities of VKOCR1v2 and vIL-6.

Expression, purification, and characterization of human mannose-6-phosphate receptor - Extra cellular domain from a stable cell line utilizing a small molecule biomimetic of the mannose-6-phosphate moiety.

The cation-independent mannose-6-phosphate receptor (CI-M6PR, aka insulin-like growth factor II receptor or IGFIIR) is a membrane protein that plays a central role in the trafficking of lysosomal acid hydrolases into lysosomes via mannose-6-phosphate (M6P) binding domains. In order to maintain cellular metabolic/catabolic homeostasis, newly synthesized lysosomal acid hydrolases are required to bind to M6PR for transit. Acid hydrolases secreted by cells can also be internalized via M6PR residing on the cell membrane and are transported to the lysosomes, a feature that enables enzyme replacement therapy for the treatment of several lysosomal storage disorders. Therefore, a thorough characterization of this receptor is critical to the development of lysosomal enzyme-based therapeutics that utilize M6PR for drug delivery to the lysosome. However, the extracellular domain (ECD) of M6PR is highly complex, containing 15-mannose receptor homology (MRH) domains. In addition, homodimerization of the receptor can occur at the membrane, making its characterization challenging. In this study, a novel human M6PR (hM6PR)-overexpressing cell line originally established for hM6PR cellular uptake assay was utilized for production of hM6PR-ECD, and a novel small molecule biomimetic (aminophenyl-M6P) affinity resin was developed for the purification of M6PR-ECD. The affinity-purified hM6PR-ECD was monomeric, contained 14 intact MRH domains (1-14) and a partial MRH domain 15, and was successfully employed in ELISA-based and surface plasmon resonance-based binding assays to demonstrate its ligand-binding functionality, making it suitable for the evaluation of biotherapeutics that utilize M6PR for cellular internalization.

Lysosomal Proteome and Secretome Analysis Identifies Missorted Enzymes and Their Nondegraded Substrates in Mucolipidosis III Mouse Cells.

Targeting of soluble lysosomal enzymes requires mannose 6-phosphate (M6P) signals whose formation is initiated by the hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α2ß2γ2). Upon proteolytic cleavage by site-1 protease, the α/ß-subunit precursor is catalytically activated but the functions of γ-subunits (Gnptg) in M6P modification of lysosomal enzymes are unknown. To investigate this, we analyzed the Gnptg expression in mouse tissues, primary cultured cells, and in Gnptg reporter mice in vivo, and found high amounts in the brain, eye, kidney, femur, vertebra and fibroblasts. Consecutively we performed comprehensive quantitative lysosomal proteome and M6P secretome analysis in fibroblasts of wild-type and Gnptgko mice mimicking the lysosomal storage disorder mucolipidosis III. Although the cleavage of the α/ß-precursor was not affected by Gnptg deficiency, the GlcNAc-1-phosphotransferase activity was significantly reduced. We purified lysosomes and identified 29 soluble lysosomal proteins by SILAC-based mass spectrometry exhibiting differential abundance in Gnptgko fibroblasts which was confirmed by Western blotting and enzymatic activity analysis for selected proteins. A subset of these lysosomal enzymes show also reduced M6P modifications, fail to reach lysosomes and are secreted, among them α-l-fucosidase and arylsulfatase B. Low levels of these enzymes correlate with the accumulation of non-degraded fucose-containing glycostructures and sulfated glycosaminoglycans in Gnptgko lysosomes. Incubation of Gnptgko fibroblasts with arylsulfatase B partially rescued glycosaminoglycan storage. Combinatorial treatments with other here identified missorted enzymes of this degradation pathway might further correct glycosaminoglycan accumulation and will provide a useful basis to reveal mechanisms of selective, Gnptg-dependent formation of M6P residues on lysosomal proteins.

Mucolipidoses Overview: Past, Present, and Future.

Mucolipidosis II and III (ML II/III) are caused by a deficiency of uridine-diphosphate N-acetylglucosamine: lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase, EC2.7.8.17), which tags lysosomal enzymes with a mannose 6-phosphate (M6P) marker for transport to the lysosome. The process is performed by a sequential two-step process: first, GlcNAc-1-phosphotransferase catalyzes the transfer of GlcNAc-1-phosphate to the selected mannose residues on lysosomal enzymes in the cis-Golgi network. The second step removes GlcNAc from lysosomal enzymes by N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (uncovering enzyme) and exposes the mannose 6-phosphate (M6P) residues in the trans-Golgi network, in which the enzymes are targeted to the lysosomes by M6Preceptors. A deficiency of GlcNAc-1-phosphotransferase causes the hypersecretion of lysosomal enzymes out of cells, resulting in a shortage of multiple lysosomal enzymes within lysosomes. Due to a lack of GlcNAc-1-phosphotransferase, the accumulation of cholesterol, phospholipids, glycosaminoglycans (GAGs), and other undegraded substrates occurs in the lysosomes. Clinically, ML II and ML III exhibit quite similar manifestations to mucopolysaccharidoses (MPSs), including specific skeletal deformities known as dysostosis multiplex and gingival hyperplasia. The life expectancy is less than 10 years in the severe type, and there is no definitive treatment for this disease. In this review, we have described the updated diagnosis and therapy on ML II/III.

Phosphosugar Stress in Bacillus subtilis: Intracellular Accumulation of Mannose 6-Phosphate Derepressed the glcR-phoC Operon from Repression by GlcR.

Bacillus subtilis phosphorylates sugars during or after their transport into the cell. Perturbation in the conversion of intracellular phosphosugars to the central carbon metabolites and accumulation of phosphosugars can impose stress on the cells. In this study, we investigated the effect of phosphosugar stress on B. subtilis Preliminary experiments indicated that the nonmetabolizable analogs of glucose were unable to impose stress on B. subtilis In contrast, deletion of manA encoding mannose 6-phosphate isomerase (responsible for conversion of mannose 6-phosphate to fructose 6-phosphate) resulted in growth arrest and bulged cell shape in the medium containing mannose. Besides, an operon encoding a repressor (GlcR) and a haloic acid dehalogenase (HAD)-like phosphatase (PhoC; previously YwpJ) were upregulated. Integration of the P glcR-lacZ cassette into different mutational backgrounds indicated that P glcR is induced when (i) a manA-deficient strain is cultured with mannose or (ii) when glcR is deleted. GlcR repressed the transcription of glcR-phoC by binding to the σA-type core elements of P glcR An electrophoretic mobility shift assay showed no interaction between mannose 6-phosphate (or other phosphosugars) and the GlcR-P glcR DNA complex. PhoC was an acid phosphatase mainly able to dephosphorylate glycerol 3-phosphate and ribose 5-phosphate. Mannose 6-phosphate was only weakly dephosphorylated by PhoC. Since deletion of glcR and phoC alone or in combination had no effect on the cells during phosphosugar stress, it is assumed that the derepression of glcR-phoC is a side effect of phosphosugar stress in B. subtilisIMPORTANCEBacillus subtilis has different stress response systems to cope with external and internal stressors. Here, we investigated how B. subtilis deals with the high intracellular concentration of phosphosugars as an internal stressor. The results indicated the derepression of an operon consisting of a repressor (GlcR) and a phosphatase (PhoC). Further analysis revealed that this operon is not a phosphosugar stress response system. The substrate specificity of PhoC may indicate a connection between the glcR-phoC operon and pathways in which glycerol 3-phosphate and ribose 5-phosphate are utilized, such as membrane biosynthesis and teichoic acid elongation.

Trypanosoma cruzi Phosphomannomutase and Guanosine Diphosphate-Mannose Pyrophosphorylase Ligandability Assessment.

Chagas' disease, which is caused by the Trypanosoma cruzi parasite, has become a global health problem that is currently treated with poorly tolerated drugs that require prolonged dosing. Therefore, there is a clinical need for new therapeutic agents that can mitigate these issues. The phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GDP-MP) enzymes form part of the de novo biosynthetic pathway to the nucleotide sugar GDP-mannose. This nucleotide sugar is used either directly, or indirectly via the formation of dolichol-phosphomannose, for the assembly of all mannose-containing glycoconjugates. In T. cruzi, mannose-containing glycoconjugates include the cell-surface glycoinositol-phospholipids and the glycosylphosphatidylinositol-anchored mucin-like glycoproteins that dominate the cell surface architectures of all life cycle stages. This makes PMM and GDP-MP potentially attractive targets for a drug discovery program against Chagas' disease. To assess the ligandability of these enzymes in T. cruzi, we have screened 18,117 structurally diverse compounds exploring drug-like chemical space and 16,845 small polar fragment compounds using an assay interrogating the activities of both PMM and GDP-MP enzymes simultaneously. This resulted in 48 small fragment hits, and on retesting 20 were found to be active against the enzymes. Deconvolution revealed that these were all inhibitors of T. cruzi GDP-MP, with compounds 2 and 3 acting as uncompetitive and competitive inhibitors, respectively. Based on these findings, the T. cruzi PMM and GDP-MP enzymes were deemed not ligandable and poorly ligandable, respectively, using small molecules from conventional drug discovery chemical space. This presents a significant hurdle to exploiting these enzymes as therapeutic targets for Chagas' disease.

Unravelling the Specificity of Laminaribiose Phosphorylase from Paenibacillus sp. YM-1 towards Donor Substrates Glucose/Mannose 1-Phosphate by Using X-ray Crystallography and Saturation Transfer Difference NMR Spectroscopy.

Glycoside phosphorylases (GPs) carry out a reversible phosphorolysis of carbohydrates into oligosaccharide acceptors and the corresponding sugar 1-phosphates. The reversibility of the reaction enables the use of GPs as biocatalysts for carbohydrate synthesis. Glycosyl hydrolase family 94 (GH94), which only comprises GPs, is one of the most studied GP families that have been used as biocatalysts for carbohydrate synthesis, in academic research and in industrial production. Understanding the mechanism of GH94 enzymes is a crucial step towards enzyme engineering to improve and expand the applications of these enzymes in synthesis. In this work with a GH94 laminaribiose phosphorylase from Paenibacillus sp. YM-1 (PsLBP), we have demonstrated an enzymatic synthesis of disaccharide 1 (ß-d-mannopyranosyl-(1→3)-d-glucopyranose) by using a natural acceptor glucose and noncognate donor substrate α-mannose 1-phosphate (Man1P). To investigate how the enzyme recognises different sugar 1-phosphates, the X-ray crystal structures of PsLBP in complex with Glc1P and Man1P have been solved, providing the first molecular detail of the recognition of a noncognate donor substrate by GPs, which revealed the importance of hydrogen bonding between the active site residues and hydroxy groups at C2, C4, and C6 of sugar 1-phosphates. Furthermore, we used saturation transfer difference NMR spectroscopy to support crystallographic studies on the sugar 1-phosphates, as well as to provide further insights into the PsLBP recognition of the acceptors and disaccharide products.

Quantitative Proteome Analysis of Mouse Liver Lysosomes Provides Evidence for Mannose 6-phosphate-independent Targeting Mechanisms of Acid Hydrolases in Mucolipidosis II.

The efficient receptor-mediated targeting of soluble lysosomal proteins to lysosomes requires the modification with mannose 6-phosphate (M6P) residues. Although the absence of M6P results in misrouting and hypersecretion of lysosomal enzymes in many cells, normal levels of lysosomal enzymes have been reported in liver of patients lacking the M6P-generating phosphotransferase (PT). The identity of lysosomal proteins depending on M6P has not yet been comprehensively analyzed. In this study we purified lysosomes from liver of PT-defective mice and 67 known soluble lysosomal proteins were identified that illustrated quantitative changes using an ion mobility-assisted data-independent label-free LC-MS approach. After validation of various differentially expressed lysosomal components by Western blotting and enzyme activity assays, the data revealed a small number of lysosomal proteins depending on M6P, including neuraminidase 1, cathepsin F, Npc2, and cathepsin L, whereas the majority reach lysosomes by alternative pathways. These data were compared with findings on cultured hepatocytes and liver sinusoid endothelial cells isolated from the liver of wild-type and PT-defective mice. Our findings show that the relative expression, targeting efficiency and lysosomal localization of lysosomal proteins tested in cultured hepatic cells resemble their proportion in isolated liver lysosomes. Hypersecretion of newly synthesized nonphosphorylated lysosomal proteins suggest that secretion-recapture mechanisms contribute to maintain major lysosomal functions in liver.

Streptomyces coelicolor strains lacking polyprenol phosphate mannose synthase and protein O-mannosyl transferase are hyper-susceptible to multiple antibiotics.

Polyprenol phosphate mannose (PPM) is a lipid-linked sugar donor used by extra-cytoplasmic glycosyl tranferases in bacteria. PPM is synthesized by polyprenol phosphate mannose synthase, Ppm1, and in most Actinobacteria is used as the sugar donor for protein O-mannosyl transferase, Pmt, in protein glycosylation. Ppm1 and Pmt have homologues in yeasts and humans, where they are required for protein O-mannosylation. Actinobacteria also use PPM for lipoglycan biosynthesis. Here we show that ppm1 mutants of Streptomyces coelicolor have increased susceptibility to a number of antibiotics that target cell wall biosynthesis. The pmt mutants also have mildly increased antibiotic susceptibilities, in particular to ß-lactams and vancomycin. Despite normal induction of the vancomycin gene cluster, vanSRJKHAX, the pmt and ppm1 mutants remained highly vancomycin sensitive indicating that the mechanism of resistance is blocked post-transcriptionally. Differential RNA expression analysis indicated that catabolic pathways were downregulated and anabolic ones upregulated in the ppm1 mutant compared to the parent or complemented strains. Of note was the increase in expression of fatty acid biosynthetic genes in the ppm1- mutant. A change in lipid composition was confirmed using Raman spectroscopy, which showed that the ppm1- mutant had a greater relative proportion of unsaturated fatty acids compared to the parent or the complemented mutant. Taken together, these data suggest that an inability to synthesize PPM (ppm1) and loss of the glycoproteome (pmt- mutant) can detrimentally affect membrane or cell envelope functions leading to loss of intrinsic and, in the case of vancomycin, acquired antibiotic resistance.

Mannose 6-phosphate-independent Lysosomal Sorting of LIMP-2.

The lysosomal integral membrane protein type 2 (LIMP-2/SCARB2) has been described as a mannose 6-phosphate (M6P)-independent trafficking receptor for ß-glucocerebrosidase (GC). Recently, a putative M6P residue in a crystal structure of a recombinantly expressed LIMP-2 ectodomain has been reported. Based on surface plasmon resonance and fluorescence lifetime imaging analyses, it was suggested that the interaction of soluble LIMP-2 with the cation-independent M6P receptor (MPR) results in M6P-dependent targeting of LIMP-2 to lysosomes. As the physiological relevance of this observation was not addressed, we investigated M6P-dependent delivery of LIMP-2 to lysosomes in murine liver and mouse embryonic fibroblasts. We demonstrate that LIMP-2 and GC reach lysosomes independent of the M6P pathway. In fibroblasts lacking either MPRs or the M6P-forming N-acetylglucosamine (GlcNAc)-1-phosphotransferase, LIMP-2 still localizes to lysosomes. Immunoblot analyses also revealed comparable LIMP-2 levels within lysosomes purified from liver of wild-type (wt) and GlcNAc-1-phosphotransferase-defective mice. Heterologous expression of the luminal domain of LIMP-2 in wild-type, LIMP-2-deficient and GlcNAc-1-phosphotransferase-defective cells further established that the M6P modification is dispensable for lysosomal sorting of LIMP-2. Finally, cathepsin Z, a known GlcNAc-1-phosphotransferase substrate, but not LIMP-2, could be precipitated with M6P-specific antibodies. These data prove M6P-independent lysosomal sorting of LIMP-2 and subsequently GC in vivo.

Lrp1/LDL Receptor Play Critical Roles in Mannose 6-Phosphate-Independent Lysosomal Enzyme Targeting.

Most lysosomal enzymes require mannose 6-phosphate (M6P) residues for efficient receptor-mediated lysosomal targeting. Although the lack of M6P residues results in missorting and hypersecretion, selected lysosomal enzymes reach normal levels in lysosomes of various cell types, suggesting the existence of M6P-independent transport routes. Here, we quantify the lysosomal proteome in M6P-deficient mouse fibroblasts (PT(ki)) using Stable Isotope Labeling by Amino acids in Cell culture (SILAC)-based comparative mass spectrometry, and find unchanged amounts of 20% of lysosomal enzymes, including cathepsins D and B (Ctsd and Ctsb). Examination of fibroblasts from a new mouse line lacking both M6P and sortilin, a candidate for M6P-independent transport of lysosomal enzymes, revealed that sortilin does not act as cargo receptor for Ctsb and Ctsd. Using fibroblast lines deficient for endocytic lipoprotein receptors, we could demonstrate that both LDL receptor and Lrp1 mediate the internalization of non-phosphorylated Ctsb and Ctsd. Furthermore, the presence of Lrp1 inhibitor increased the secretion of Ctsd from PT(ki) cells. These findings establish Lrp1 and LDL receptors in M6P-independent secretion-recapture targeting mechanism for lysosomal enzymes.

Multiple Domains of GlcNAc-1-phosphotransferase Mediate Recognition of Lysosomal Enzymes.

The Golgi enzyme UDP-GlcNAc:lysosomal enzymeN-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), an α2ß2γ2hexamer, mediates the initial step in the addition of the mannose 6-phosphate targeting signal on newly synthesized lysosomal enzymes. This tag serves to direct the lysosomal enzymes to lysosomes. A key property of GlcNAc-1-phosphotransferase is its unique ability to distinguish the 60 or so lysosomal enzymes from the numerous non-lysosomal glycoproteins with identical Asn-linked glycans. In this study, we demonstrate that the two Notch repeat modules and the DNA methyltransferase-associated protein interaction domain of the α subunit are key components of this recognition process. Importantly, different combinations of these domains are involved in binding to individual lysosomal enzymes. This study also identifies the γ-binding site on the α subunit and demonstrates that in the majority of instances the mannose 6-phosphate receptor homology domain of the γ subunit is required for optimal phosphorylation. These findings serve to explain how GlcNAc-1-phosphotransferase recognizes a large number of proteins that lack a common structural motif.

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

Targeting soluble acid hydrolases to lysosomes requires the addition of mannose 6-phosphate residues on their N-glycans. This process is initiated by GlcNAc-1-phosphotransferase, a multi-subunit enzyme encoded by the GNPTAB and GNPTG genes. The GNPTAB gene products (the α and ß subunits) are responsible for recognition and catalysis of hydrolases whereas the GNPTG gene product (the γ subunit) enhances mannose phosphorylation of a subset of hydrolases. Here we identify and characterize a zebrafish gnptg insertional mutant and show that loss of the gamma subunit reduces mannose phosphorylation on a subset glycosidases but does not affect modification of several cathepsin proteases. We further show that glycosidases, but not cathepsins, are hypersecreted from gnptg(-/-) embryonic cells, as evidenced by reduced intracellular activity and increased circulating serum activity. The gnptg(-/-) embryos lack the gross morphological or craniofacial phenotypes shown in gnptab-deficient morphant embryos to result from altered cathepsin activity. Despite the lack of overt phenotypes, decreased fertilization and embryo survival were noted in mutants, suggesting that gnptg associated deposition of mannose 6-phosphate modified hydrolases into oocytes is important for early embryonic development. Collectively, these findings demonstrate that loss of the zebrafish GlcNAc-1-phosphotransferase γ subunit causes enzyme-specific effects on mannose phosphorylation. The finding that cathepsins are normally modified in gnptg(-/-) embryos is consistent with data from gnptab-deficient zebrafish suggesting these proteases are the key mediators of acute pathogenesis. This work also establishes a valuable new model that can be used to probe the functional relevance of GNPTG mutations in the context of a whole animal.