Dolichol Phosphate Mannose Synthase Research Articles

DPM2 serve as novel oncogene and prognostic marker transactivated by ESR1 in breast cancer.

Breast cancer (BRCA) is the most common malignancies worldwide with increasing rate. Dolichol phosphate mannose synthase (DPMS) is a critical mannosyltransferase involved in the posttranslational modification of proteins. At present, there is limited knowledge regarding the function of DPMS in breast cancer. In this study, silica analysis in multiple datasets found that dolichyl-phosphate mannosyltransferase subunit 2 (DPM2) is an unfavorable prognostic marker, suggesting its oncogenic role. Cell counting kit-8 and apoptosis assays show that DPM2-silenced cancer cells exhibit decreased growth potential and enhanced cell death rate. Further, transwell and wound healing assays show reduced invasion and migration capabilities in DPM2 knockdown groups, xenograft nude mice model demonstrated smaller tumor volume in DPM2 silenced BC cells. Then, the underlying downstream mechanism of DPM2 in BC was predicted and analyzed, highlighting classical tumorigenic pathways like JAK/STAT signaling pathway and oxidative phosphorylation activated in the cancer group. Finally, ChIP-seq analysis, expression correlation analysis, inhibitor treatment, and dual luciferase assays show that DPM2 is transcriptionally activated by estrogen receptor1 (ESR1). The results show that high expression of DPM2 mRNA is significantly correlated with shorter overall survival (OS) and disease-free survival (DFS) in breast cancer patients, and in vitro knockdown of DPM2 can significantly inhibit the malignant phenotypes of cells, including proliferation, invasion, migration, and apoptosis. These results suggest that DPM2 may play an important role in breast cancer. Altogether, we first uncovered the tumorigenic and prognostic role of DPM2 in breast cancer, cellular assays, and bioinformatics analysis highlighted DPM2 as oncogene via inhibited cancer-related signaling pathways in breast cancer. Besides, DPM2 is transcriptionally activated by ESR1, the signaling axis of ESR1/DPM2 provides a new strategy for BC-targeted therapy.

Identification and characterization of a new variation in DPM2 gene in two Chinese siblings with mild intellectual impairment.

Introduction: Congenital disorders of glycosylation (CDGs) are a genetically heterogeneous group of metabolic disorders caused by abnormal protein or lpid glycosylation. DPM2 is one subunit of a heterotrimeric complex for dolichol-phosphatemannose synthase (DPMS), a key enzyme in glycosylation, and only four patients with DPM2-CDG have been reported. Methods: Whole-exome sequencing (WES) was performed in a Chinese family having two siblings with a mild form of DPM2-CDG with developmental delay, mild intellectual disability, hypotonia, and increased serum creatine kinase. Sanger sequencing was used to validate the variants identified in the siblings and their parents. In vitro functional study was performed. Results: A homozygous mutation, c.197G>A (p.Gly66Glu) in exon 4 of DPM2 (NM_003863) was identified by whole exome sequencing (WES). In vitro functional analysis demonstrated that this variant increased the expression level of DPM2 protein and western blot revealed a significant decrease in ICAM1, a universal biomarker for hypoglycosylation in patients with CDG, suggesting abnormal N-linked glycosylation. We also reviewed the 4 previously reported patients carrying homozygous or compound heterozygous variants of DMP2 gene, and found that patients with variants within the region encoding the first domain had more severe clinical symptoms than those with variants within the second domain. However, the actual genotype-phenotype relationship needs more study. Discussion: Overall, our study broadens the variant spectrum of DPM2 gene, attempts to explain the different phenotypes in patients with different DPM2 variants, and emphasizes the need of further functional studies to understand the underlying pathophysiology of the phenotypic heterogeneity.

A recurrent homozygous missense DPM3 variant leads to muscle and brain disease.

Biallelic pathogenic variants in the genes encoding the dolichol-phosphate mannose synthase subunits (DPM) which produce mannosyl donors for glycosylphosphatidylinositols, N-glycan and protein O- and C-mannosylation, are rare causes of congenital disorders of glycosylation. Pathogenic variants in DPM1 and DPM2 are associated with muscle-eye-brain (MEB) disease, whereas DPM3 variants have mostly been reported in patients with isolated muscle disease-dystroglycanopathy. Thus far, only one affected individual with compound heterozygous DPM3 variants presenting with myopathy, mild intellectual disability, seizures, and nonspecific white matter abnormalities (WMA) around the lateral ventricles has been described. Here we present five affected individuals from four unrelated families with global developmental delay/intellectual disability ranging from mild to severe, microcephaly, seizures, WMA, muscle weakness and variable cardiomyopathy. Exome sequencing of the probands revealed an ultra-rare homozygous pathogenic missense DPM3 variant NM_018973.4:c.221A>G, p.(Tyr74Cys) which segregated with the phenotype in all families. Haplotype analysis indicated that the variant arose independently in three families. Functional analysis did not reveal any alteration in the N-glycosylation pathway caused by the variant; however, this does not exclude its pathogenicity in the function of the DPM complex and related cellular pathways. This report provides supporting evidence that, besides DPM1 and DPM2, defects in DPM3 can also lead to a muscle and brain phenotype.

Aspirin damages the cell wall of Saccharomyces cerevisiae by inhibiting the expression and activity of dolichol-phosphate mannose synthase 1.

Aspirin is a commonly used anti-inflammatory, analgesic and antithrombotic drug. It has attracted attention due to its potential antifungal therapeutic effect; however, the molecular mechanism is poorly understood. Here, the effects of aspirin on the cell wall of Saccharomyces cerevisiae were explored. We observed by scanning electron microscopy that aspirin could damage the cell wall ultrastructure. Meanwhile, a cellular surface hydrophobicity (CSH) assay showed that aspirin increased the hydrophobicity of the yeast cell surface. A drug sensitivity assay indicated that the overexpression of dolichol phosphate mannose synthase 1 (DPM1) reversed the cell wall damage and decreased the CSH induced by aspirin. Importantly, aspirin decreased the expression and enzyme activity of DPM1 in S. cerevisiae. Molecular docking results demonstrated that aspirin could directly bind to the Ser141 site of DPM1. Similarly, we found that aspirin damaged the cell wall and inhibited the expression of DPM1 in Candida albicans. These findings improve the current understanding of the action mode of aspirin and provide new strategies for antifungal drug design.

Dissection and enhancement of prebiotic properties of yeast cell wall oligosaccharides through metabolic engineering

Saccharomyces boulardii is a yeast clinically used for treating various symptoms of gastrointestinal dysbiosis. Despite their genomic relatedness, S. boulardii has a distinctive cell wall oligosaccharide composition compared to baker's yeast S. cerevisiae, such as higher mannan content. Here we explore the beneficial effects of S. boulardii cell wall oligosaccharides through metabolic engineering. We increased the production of guanosine diphosphate (GDP)-mannose, the substrate for cell wall mannan biosynthesis, by perturbing glycolysis flux and overexpressing the enzymes in the GDP-mannose biosynthesis pathway. Combined with overexpression of a cell wall mannoprotein and dolichol phosphate mannose synthase, the cell wall mannan content of S. boulardii increased up to 52%. The identical engineering resulted in marginal changes in the S. cerevisiae cell wall. S. boulardii showed a higher adhesive capacity against Salmonella enterica Typhimurium than S. cerevisiae, and yeast-bacteria sedimentation rates were positively correlated with cell wall mannan contents. Besides, S. boulardii biomass selectively proliferated Bacteroides thetaiotaomicron over Clostridioides difficile more efficiently than S. cerevisiae, and the selectivity was further enhanced by amplifying the cell wall mannan. Collectively, we report the important prebiotic roles of cell wall oligosaccharides in the protective functions of S. boulardii and present a unique metabolic engineering approach to modulate the functions.

D-Allulose Production from d-fructose by Putative Dolichol Phosphate Mannose Synthase from Bacillus sp. with Potential d-allulose 3-epimrase Activity

d-Allulose Production from d-fructose by Putative Dolichol Phosphate Mannose Synthase from Bacillus sp. with Potential d-allulose 3-epimrase Activity

Whole-Genome CRISPR Screening Identifies N-Glycosylation As an Essential Pathway and a Potential Novel Therapeutic Target in CALR-Mutant MPN

Somatic mutations in the ER chaperone calreticulin (CALR) are frequent and disease-initiating in myeloproliferative neoplasms (MPN). Although the mechanism of mutant CALR-induced MPN is known to involve pathogenic binding between mutant CALR and MPL, this insight has not yet been exploited therapeutically. Consequently, a major deficiency is the lack of clonally selective therapeutic agents with curative potential. Hence, we set out to discover and validate unique genetic dependencies for mutant CALR-driven oncogenesis.We first performed a whole-genome CRISPR knockout screen in CALR Δ52 MPL-expressing hematopoietic cells to identify genes that were differentially required for the growth of cytokine-independent, transformed CALR Δ52 cells as compared to control cells. Using gene-set enrichment analyses, we identified the N-glycan biosynthesis, unfolded protein response, and the protein secretion pathways to be amongst the most significantly differentially depleted pathways (FDR q values <0.001, 0.014, and 0.025, respectively) in CALR Δ52 cells. We performed a secondary CRISPR pooled screen focused on significant pathways from the primary screen and confirmed these findings.Strikingly, seven of the top ten hits in both screens were linked to protein N-glycosylation. Four of those genes encode proteins involved in the enzymatic activity of dolichol-phosphate mannose synthase (DPM1, DPM2, DPM3, and MPDU1). This enzyme synthesizes dolichol D-mannosyl phosphate, an essential substrate for protein N-glycosylation. Importantly, these findings from an unbiased whole-genome screen align with prior mechanistic studies demonstrating that both the N-glycosylation sites on MPL and the lectin-binding sites on CALR Δ52 are required for mutant CALR-driven oncogenesis.We next performed single gene CRISPR Cas9 validation studies and found that DPM2 is required for CALR Δ52-mediated transformation, as demonstrated by increased cell death, reduced p-STAT5 and decreased MPL cell-surface levels, when Dpm2 is knocked out. Importantly, cells cultured in cytokine-rich medium were unaffected by DPM2 loss. Upon cytokine withdrawal, a sub-clone of non-edited Dpm2WT CALR Δ52 cells grew out, further demonstrating requirement for DPM2 for the survival of CALR Δ52 cells. Additionally, we observed a >50% reduction in ex vivo myeloid colony formation of murine CalrΔ52 Dpm2 ko bone marrow (BM) compared with CRISPR-Cas9 non-targeting controls, with non-significant effects on CalrWT BM cells.To enable clinical translation, we performed a pharmacological screen targeting pathways significantly depleted in our CRISPR screens. Screening 70 drugs, we found that the N-glycosylation pathway was the only pathway in which all tested compounds preferentially killed CALR Δ52 transformed cells.We then treated primary Calr Δ52/+ mice with a clinical grade N-glycosylation (N-Gi) inhibitor and found platelet counts (Sysmex) to be significantly reduced (vehicle 3x10 6/mL, N-Gi 1x10 6/mL after 18 days, p<.0001). Concordantly, the proportion of megakaryocyte erythrocyte progenitors (MEPs) was significantly reduced in CalrΔ52 BM (p=0.03).We next performed competitive BM transplantation assays using CD45.2 UBC-GFP MxCre CalrΔ52 knockin and CD45.1 mice. We found that mice treated with N-Gi had significantly reduced platelet counts (vehicle 1440x10 6/mL, N-Gi 845x10 6/mL, p=0.005) as well as significantly reduced platelet chimerism (vehicle 55%, N-Gi 27%, p<0.001), indicating a distinct vulnerability of CalrΔ52 over WT cells.Finally, we interrogated RNA-sequencing data from primary human MPN platelets. We found N-glycosylation-related pathways to be significantly upregulated in CALR-mutated platelets (n = 13) compared to healthy control platelets (n = 21), highlighting the relevance of our findings to human MPN.In summary, using unbiased genetic and focused pharmacological screens, we identified the N-glycan biosynthesis pathway as essential for mutant CALR-driven oncogenesis. Using a pre-clinical MPN model, we found that in vivo inhibition of N-glycosylation normalizes key features of MPN and preferentially targets CalrΔ52 over WT cells. These findings have therapeutic implications through inhibiting N-glycosylation alone or in combination with other agents to advance the development of clonally selective therapeutic approaches in CALR-mutant MPN.AEM and JSJ contributed equally. [Display omitted] DisclosuresMullally: Janssen, PharmaEssentia, Constellation and Relay Therapeutics: Consultancy.

Distinct Regions of the Haloferax volcanii Dolichol Phosphate-Mannose Synthase AglD Mediate the Assembly and Subsequent Processing of the Lipid-Linked Mannose.

Haloferax volcanii AglD is currently the only archaeal dolichol phosphate (DolP)-mannose synthase shown to participate in N-glycosylation. However, the relation between AglD and Pyrococcus furiosus PF0058, the only archaeal DolP-mannose synthase for which structural information is presently available, was unclear. In this report, similarities between the PF0058 and AglD catalytic domains were revealed. At the same time, AglD includes a transmembrane domain far longer than that of PF0058 or other DolP-mannose synthases. To determine whether this extension affords AglD functions in addition to generating mannose-charged DolP, a series of Hfx. volcanii strains expressing truncated versions of AglD was generated. Mass spectrometry revealed that a version of AglD comprising the catalytic domain and only two of the six to nine predicted membrane-spanning domains could mediate mannose addition to DolP. However, in cells expressing this or other truncated versions of AglD, mannose was not transferred from the lipid to the protein-bound tetrasaccharide precursor of the N-linked pentasaccharide normally decorating Hfx. volcanii glycoproteins. These results thus point to AglD as contributing to additional aspects of Hfx. volcanii N-glycosylation beyond charging DolP with mannose. Accordingly, the possibility that AglD, possibly in coordination with AglR, translocates DolP-mannose across the plasma membrane is discussed.

DPM1 expression as a potential prognostic tumor marker in hepatocellular carcinoma.

BackgroundAltered glycosylation of proteins contributes to tumor progression. Dolichol phosphate mannose synthase (DPMS), an essential mannosyltransferase, plays a central role in post-translational modification of proteins, including N-linked glycoproteins, O-mannosylation, C-mannosylation and glycosylphosphatidylinositol anchors synthesis. Little is known about the function of DPMS in liver cancer.MethodsThe study explored the roles of DPMS in the prognosis of hepatocellular carcinoma using UALCAN, Human Protein Atlas, GEPIA, cBioPortal and Metascape databases. The mRNA expressions of DPM1/2/3 also were detected by quantitative real-time PCR experiments in vitro.ResultsThe transcriptional and proteinic expressions of DPM1/2/3 were both over-expressed in patients with hepatocellular carcinoma. Over-expressions of DPMS were discovered to be dramatically associated with clinical cancer stages and pathological tumor grades in hepatocellular carcinoma patients. In addition, higher mRNA expressions of DPM1/2/3 were found to be significantly related to shorter overall survival in liver cancer patients. Futhermore, high genetic alteration rate of DPMS (41%) was also observed in patients with liver cancer, and genetic alteration in DPMS was associated with shorter overall survival in hepatocellular carcinoma patients. We also performed quantitative real-time PCR experiments in human normal hepatocytes and hepatoma cells to verify the expressions of DPM1/2/3 and results showed that the expression of DPM1 was significantly increased in hepatoma cells SMMC-7721 and HepG2.ConclusionsTaken together, these results suggested that DPM1 could be a potential prognostic biomarker for survivals of hepatocellular carcinoma patients.

Retinal Degeneration Caused by Rod-Specific Dhdds Ablation Occurs without Concomitant Inhibition of Protein N-Glycosylation.

SummaryDehydrodolichyl diphosphate synthase (DHDDS) catalyzes the committed step in dolichol synthesis. Recessive mutations in DHDDS cause retinitis pigmentosa (RP59), resulting in blindness. We hypothesized that rod photoreceptor-specific ablation of Dhdds would cause retinal degeneration due to diminished dolichol-dependent protein N-glycosylation. Dhddsflx/flx mice were crossed with rod-specific Cre recombinase-expressing (Rho-iCre75) mice to generate rod-specific Dhdds knockout mice (Dhddsflx/flx iCre+). In vivo morphological and electrophysiological evaluation of Dhddsflx/flx iCre+ retinas revealed mild retinal dysfunction at postnatal (PN) 4 weeks, compared with age-matched controls; however, rapid photoreceptor degeneration ensued, resulting in almost complete loss of rods and cones by PN 6 weeks. Retina dolichol levels were markedly decreased by PN 4 weeks in Dhddsflx/flx iCre+ mice, relative to controls; despite this, N-glycosylation of retinal proteins, including opsin (the dominant rod-specific glycoprotein), persisted in Dhddsflx/flx iCre+ mice. These findings challenge the conventional mechanistic view of RP59 as a congenital disorder of glycosylation.

DPMS is Essential for Tumor Angiogenesis

Dolichol phosphate mannose synthase (DPMS) an inverting GT‐A folded enzyme and classified as GT2 by CAZy (carbohydrate active enZyme; http://www.cazy.org). It catalyzes the transfer reaction Dol‐P + GDP‐Man &lt; == &gt; Dol‐P‐Man + GDP in endoplasmic reticulum (ER) for the synthesis of dolichol‐linked tetra‐decasaccharide [Glc3Man9GlcNAc2‐PP‐Dol; lipid linked oligosaccharide (LLO)], a pre‐requisite for the glycosylation of asparagine‐linked (N‐linked) glycoproteins. N‐Glycosylation provides structural and functional stability, and adds flexibility to glycoproteins. The gene for DPMS has been cloned from many species, i.e., from archaea to human. The amino acid sequence identifies the presence of a metal binding (i.e., DAD) signature and a cAMP‐dependent protein phosphorylation (PKA) motif. Phosphorylation enhances the DPMS catalytic activity and consequently increases the synthesis and turnover of LLO. In capillary endothelial cells, it means increased proliferation, i.e., angiogenesis. Angiogenesis is a hallmark for breast cancer progression. Therefore, we have hypothesized that overexpression of DPMS in capillary endothelial cells could mimic the cAMP signaling and independently increase angiogenesis. To test the hypothesis, we have used a cell line that stably overexpresses DPMS and evaluated the role of D(+)mannose on their proliferation by MTT assay. Capillary endothelial cells harboring the DPMS overexpressing plasmid exhibited increased expression of DPMS when examined by immunofluorescence microscopy, western blotting, and QPCR. There was increased expression of cell surface N‐glycans as judged by Texas‐Red conjugated WGA‐binding. The cell proliferation as well as the cellular mitotic activity were also enhanced. To evaluate if mannose is required for the observed effect, we have monitored the cellular proliferation as a function of mannose concentration and the time of incubation by MTT assay. The results indicate mannose supplementation did not support the growth of neither the DPMS overexpressing cells nor the control cells. Triple negative human breast cancer cells also showed a similar result. We therefore conclude DPMS overexpression induces angiogenesis. Mechanistically it is not due to upregulation of mannose transport. The environmental factors increasing intracellular cAMP level increase DPMS catalytic activity metabolically or through gene expression and enhance angiogenesis for breast tumor progression. DPMS, thus could be an excellent target for developing a new generation glycotherapy against breast cancer.Support or Funding InformationInstitutionalThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dolichol Phosphate Mannose Synthase: A Glycosyltransferase with Unity in Molecular Diversities

N‐glycans provide structural and functional stability to asparagine‐linked (N‐linked) glycoprotein, and add flexibility. Glycan biosynthesis is elaborative, multi‐compartmental and involves many glycosyltransferases. Failure to assemble N‐glycans leads to phenotypic changes developing infection, cancer, congenital disorders of glycosylation (CDGs) among others. Biosynthesis of N‐glycans begins at the endoplasmic reticulum (ER) with the assembly of dolichol‐linked tetra‐decasaccharide (Glc3Man9GlcNAc2‐PP‐Dol) where dolichol phosphate mannose synthase (DPMS) plays a central role. DPMS is also essential for GPI anchor biosynthesis as well as for O‐ and C‐mannosylation of proteins in yeast and in mammalian cells. DPMS has been purified from several sources and its gene has been cloned from 39 species (e.g., from protozoan parasite to human). It is an inverting GT‐A folded enzyme and classified as GT2 by CAZy (carbohydrate active enZyme; http://www.cazy.org). The sequence alignment detects the presence of a metal binding DAD signature in DPMS from all 39 species but finds cAMP‐dependent protein phosphorylation motif (PKA motif) in only 38 species. DPMS also has hydrophobic region(s). Hydropathy analysis of amino acid sequences from bovine, human, S. crevisiae and A. thaliana DPMS show PKA motif is present between the hydrophobic domains. The location of PKA motif as well as the hydrophobic domain(s) in the DPMS sequence vary from species to species. For example, the domain(s) could be located at the center or more towards the C‐terminus. Irrespective of their catalytic similarity, the DNA sequence, the amino acid identity, and the lack of a stretch of hydrophobic amino acid residues at the C‐terminus, DPMS has been classified as Type I and Type II enzyme. DPMS belonging to Type II sub‐class has been considered to be a multimeric protein requiring the DPM2 and DPM3 to anchor down the DPM1 to the ER membrane. Our results indicate that the recombinant DPMS from bovine adrenal medullary capillary endothelial cells can be expressed successfully in E. coli as an active enzyme. Furthermore, DPMS is regulated by extracellular signaling and microenvironment. So, we conclude that it is the hydrophobic domain that anchor down DPMS to the ER membrane.Support or Funding InformationSupported in part by the grant NIH/NIMHD G12MD007583 (KB)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dynamic Function of DPMS Is Essential for Angiogenesis and Cancer Progression.

Dolichol phosphate mannose synthase (DPMS) is an inverting GT-A-folded enzyme and classified as GT2 by CAZy. DPMS sequence carries a metal-binding DXD motif, a PKA motif, and a variable number of hydrophobic domains. Human and bovine DPMS possess a single transmembrane domain, whereas that from S. cerevisiae and A. thaliana carry multiple transmembrane domains and are superimposable. The catalytic activity of DPMS is documented in all spheres of life, and the 32kDa protein is uniquely regulated by protein phosphorylation. Intracellular activation of DPMS by cAMP signaling is truly due to the activation of the enzyme and not due to increased Dol-P level. The sequence of DPMS in some species also carries a protein N-glycosylation motif (Asn-X-Ser/Thr). Apart from participating in N-glycan biosynthesis, DPMS is essential for the synthesis of GPI anchor as well as for O- and C-mannosylation of proteins. Because of the dynamic nature, DPMS actively participates in cellular proliferation enhancing angiogenesis and breast tumor progression. In fact, overexpression of DPMS in capillary endothelial cells supports increased N-glycosylation, cellular proliferation, and enhanced chemotactic activity. These are expected to be completely absent in congenital disorders of glycosylation (CDGs) due to the silence of DPMS catalytic activity. DPMS has also been found to be involved in the cross talk with N-acetylglucosaminyl 1-phosphate transferase (GPT). Inhibition of GPT with tunicamycin downregulates the DPMS catalytic activity quantitatively. The result is impairment of surface N-glycan expression, inhibition of angiogenesis, proliferation of human breast cancer cells, and induction of apoptosis. Interestingly, nano-formulated tunicamycin is three times more potent in inhibiting the cell cycle progression than the native tunicamycin and is supported by downregulation of the ratio of phospho-p53 to total-p53 as well as phospho-Rb to total Rb. DPMS expression is also reduced significantly. However, nano-formulated tunicamycin does not induce apoptosis. We, therefore, conclude that DPMS could become a novel target for developing glycotherapy treating breast tumor in the clinic.

Dolichol phosphate mannose synthase: a Glycosyltransferase with Unity in molecular diversities.

N-glycans provide structural and functional stability to asparagine-linked (N-linked) glycoproteins, and add flexibility. Glycan biosynthesis is elaborative, multi-compartmental and involves many glycosyltransferases. Failure to assemble N-glycans leads to phenotypic changes developing infection, cancer, congenital disorders of glycosylation (CDGs) among others. Biosynthesis of N-glycans begins at the endoplasmic reticulum (ER) with the assembly of dolichol-linked tetra-decasaccharide (Glc3Man9GlcNAc2-PP-Dol) where dolichol phosphate mannose synthase (DPMS) plays a central role. DPMS is also essential for GPI anchor biosynthesis as well as for O- and C-mannosylation of proteins in yeast and in mammalian cells. DPMS has been purified from several sources and its gene has been cloned from 39 species (e.g., from protozoan parasite to human). It is an inverting GT-A folded enzyme and classified as GT2 by CAZy (carbohydrate active enZyme; http://www.cazy.org ). The sequence alignment detects the presence of a metal binding DAD signature in DPMS from all 39 species but finds cAMP-dependent protein phosphorylation motif (PKA motif) in only 38 species. DPMS also has hydrophobic region(s). Hydropathy analysis of amino acid sequences from bovine, human, S. crevisiae and A. thaliana DPMS show PKA motif is present between the hydrophobic domains. The location of PKA motif as well as the hydrophobic domain(s) in the DPMS sequence vary from species to species. For example, the domain(s) could be located at the center or more towards the C-terminus. Irrespective of their catalytic similarity, the DNA sequence, the amino acid identity, and the lack of a stretch of hydrophobic amino acid residues at the C-terminus, DPMS is still classified as Type I and Type II enzyme. Because of an apparent bio-sensing ability, extracellular signaling and microenvironment regulate DPMS catalytic activity. In this review, we highlight some important features and the molecular diversities of DPMS.

Dolichol-phosphate mannose synthase depletion in zebrafish leads to dystrophic muscle with hypoglycosylated α-dystroglycan

Defective dolichol-phosphate mannose synthase (DPMS) complex is a rare cause of congenital muscular dystrophy associated with hypoglycosylation of alpha-dystroglycan (α-DG) in skeletal muscle. We used the zebrafish (Danio rerio) to model muscle abnormalities due to defects in the subunits of DPMS. The three zebrafish ortholog subunits (encoded by the dpm1, dpm2 and dpm3 genes, respectively) showed high similarity to the human proteins, and their expression displayed localization in the midbrain/hindbrain area and somites. Antisense morpholino oligonucleotides targeting each subunit were used to transiently deplete the dpm genes. The resulting morphant embryos showed early death, muscle disorganization, low DPMS complex activity, and increased levels of apoptotic nuclei, together with hypoglycosylated α-DG in muscle fibers, thus recapitulating most of the characteristics seen in patients with mutations in DPMS. Our results in zebrafish suggest that DPMS plays a role in stabilizing muscle structures and in apoptotic cell death.

Structure of the polyisoprenyl-phosphate glycosyltransferase GtrB and insights into the mechanism of catalysis.

The attachment of a sugar to a hydrophobic polyisoprenyl carrier is the first step for all extracellular glycosylation processes. The enzymes that perform these reactions, polyisoprenyl-glycosyltransferases (PI-GTs) include dolichol phosphate mannose synthase (DPMS), which generates the mannose donor for glycosylation in the endoplasmic reticulum. Here we report the 3.0Å resolution crystal structure of GtrB, a glucose-specific PI-GT from Synechocystis, showing a tetramer in which each protomer contributes two helices to a membrane-spanning bundle. The active site is 15 Å from the membrane, raising the question of how water-soluble and membrane-embedded substrates are brought into apposition for catalysis. A conserved juxtamembrane domain harbours disease mutations, which compromised activity in GtrB in vitro and in human DPM1 tested in zebrafish. We hypothesize a role of this domain in shielding the polyisoprenyl-phosphate for transport to the active site. Our results reveal the basis of PI-GT function, and provide a potential molecular explanation for DPM1-related disease.

Dolichol phosphate mannose synthase from the pathogenic yeast Candida albicans is a multimeric enzyme

BackgroundDolichol phosphate mannose synthase (DPMS) is a key enzyme in N- and O-linked glycosylations and glycosylphosphatidylinositol (GPI)-anchor synthesis. DPMS generates DPM, the substrate for mentioned processes, by the transfer of mannosyl residue from GDP-Man to dolichol phosphate. Here we describe the role of DPMS for Candida albicans physiology with emphasis on the cell wall composition and morphogenesis. MethodsC. albicans genes for DPMS subunits were cloned, tagged and expressed in Saccharomyces cerevisiae. The C. albicans strains with controlled expression of DPM genes were constructed and analyzed. Gene expression and enzyme activities were measured using RT-PCR and radioactive substrate. Sensitivities against chemical agents were tested with microdilution method. The composition of the cell wall was estimated by HPLC. Glycosylation status of the marker protein was analyzed by Western blot. Morphological differentiation of the strains was checked on the media promoting hyphae and chlamydospore formation. ResultsWe demonstrate that C. albicans DPMS consists of three interacting subunits, among which Dpm1 and Dpm3 are indispensable, whereas Dpm2 increases enzymatic activity. Lowered expression of DPMS genes results in decreased DPMS activity, increased susceptibility to cell wall perturbing agents and in altered cell wall composition. Mutants Tetp-DPM1 and Tetp-DPM3 show defective protein glycosylation and are impaired in hyphae and chlamydospore formation. Major conclusionDPMS from C. albicans, opposite to S. cerevisiae, belongs to the family of DPMS with multimeric protein structure. General significanceThis work provides important data about factors required for a proper protein glycosylation and for morphogenesis of pathogenic yeast C. albicans.

Glyco‐engineering in Archaea: differential N‐glycosylation of the S‐layer glycoprotein in a transformed Haloferax volcanii strain

SummaryArchaeal glycoproteins present a variety of N‐linked glycans not seen elsewhere. The ability to harness the agents responsible for this unparalleled diversity offers the possibility of generating glycoproteins bearing tailored glycans, optimized for specific functions. With a well‐defined N‐glycosylation pathway and available genetic tools, the haloarchaeon Haloferax volcanii represents a suitable platform for such glyco‐engineering efforts. In Hfx. volcanii, the S‐layer glycoprotein is modified by an N‐linked pentasaccharide. In the following, S‐layer glycoprotein N‐glycosylation was considered in cells in which AglD, the dolichol phosphate mannose synthase involved in addition of the final residue of the pentasaccharide, was replaced by a haloarchaeal homologue of AglJ, the enzyme involved in addition of the first residue of the N‐linked pentasaccharide. In the engineering strain, the S‐layer glycoprotein is modified by a novel N‐linked glycan not found on this reporter from the parent strain. Moreover, deletion of AglD alone and introduction of the AglJ homologue from Halobacterium salinarum, OE2528R, into the deletion strain resulted in increased biosynthesis of the novel 894 Da glycan concomitant with reduced biogenesis of the pentasaccharide normally N‐linked to the S‐layer glycoprotein. These findings justify efforts designed to transform Hfx. volcanii into a glyco‐engineering ‘workshop’.

Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells

Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man 5GlcNAc 2-P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man 9GlcNAc 2-P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc 3Man 9GlcNAc 2-P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man 5GlcNAc 2-PP-Dol through Glc 1Man 9GlcNAc 2-PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc 3Man 9GlcNAc 2-P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

Protein Glycosylation in Candida

Candidiasis is a significant cause of invasive human mycosis with associated mortality rates that are equivalent to, or worse than, those cited for most cases of bacterial septicemia. As a result, considerable efforts are being made to understand how the fungus invades host cells and to identify new targets for fungal chemotherapy. This has led to an increasing interest in Candida glycobiology, with an emphasis on the identification of enzymes essential for glycoprotein and adhesion metabolism, and the role of N- and O-linked glycans in host recognition and virulence. Here, we refer to studies dealing with the identification and characterization of enzymes such as dolichol phosphate mannose synthase, dolichol phosphate glucose synthase and processing glycosidases and synthesis, structure and recognition of mannans and discuss recent findings in the context of Candida albicans pathogenesis.