Mannosylphosphodolichol synthase overexpression supports angiogenesis

Abstract

Mannosylphosphodolichol synthase (DPMS) plays a critical role in Glc₃Man₉GlcNAc₂-PP-Dol (lipid-linked oligosaccha-ride, LLO) biosynthesis, an essential intermediate in asparagine-linked (N-linked) protein glycosylation. We observed earlier that phosphorylation of DPMS increases the catalytic activity of the enzyme by increasing the V_max as well as the enzyme turnover (k_cat) without significantly changing the K_m for GDP-mannose. As a result, LLO biosynthesis, turnover and protein N-glycosylation are increased. This is manifested in increased proliferation of capillary endothelial cells, i.e. angiogenesis. We have then asked, if the phosphorylation event or the upregulation of DPMS due to overproduction of the enzyme is the key factor in upregulating angiogenesis? This question has been answered by isolating a stable capillary endothelial cell clone overexpressing the gene encoding DPMS. Our results indicate that the DPMS-overexpressing clone has a high level of DPMS mRNA as judged by QRT-PCR. The clone also expresses nearly four times more DPMS protein than the clone transfected with pEGFP-N1 vector only (i.e. control) as analyzed by Western blotting. Most importantly, the overexpressing DPMS clone has ～108% higher DPMS activity than the vector control. Immunofluorescence microscopy with Texas Red-conjugated wheat germ agglutinin indicates a high level of expression of (GlcNAc-β-(1,4)-GlcNAc) 1-4-β-GlcNAc-NeuAc glycans on the external surface of the capillary endothelial cells overexpressing DPMS. Increased cellular proliferation and accelerated healing of the wound induced by mechanical stress of the DPMS-overexpressing clone unequivocally supports a role of DPMS in angiogenesis.     

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.     

Cloning and characterization of an mRNA encoding a novel G protein α-subunit abundant in mantle and gill of pearl oyster Pinctada fucata

Nacre formation is an ideal model to study biomineralization processes. Although much has been done about biomineralization mechanism of nacre, little is known as to how cellular signaling regulates this process. We are interested in whether G protein signaling plays a role in mineralization. Degenerate primers against conserved amino acid regions of G proteins were employed to amplify cDNA from the pearl oyster Pinctada fucata. As a result, the cDNA encoding a novel G_sα (pfG_sα) from the pearl oyster was isolated. The G_sα cDNA encodes a polypeptide of 377 amino acid residues, which shares high similarity to the octopus (Octopus vulgaris) G_sα. The well-conserved A, C, G (switch I), switch II functional domains and the carboxyl terminus that is a critical site for interaction with receptors are completely identical to those from other mollusks. However, pfG_sα has a unique amino acid sequence, which encodes switch III and interaction sites of adenylyl cyclase respectively. In situ hybridization and Northern blotting analysis revealed that the oyster G_sα mRNA is widely expressed in a variety of tissues, with highest levels in the outer fold of mantle and epithelia of gill, the regions essential for biomineralization. We also show that overexpression of the pfG_sα in mammalian MC3T3-E1 cells resulted in increased cAMP levels. Mutant pfG_sα that has impaired CTX substrate diminished its ability to induce cAMP production. Furthermore, the alkaline phosphatase (ALP) activity, an indicator for mineralization, is induced by the G_sα in MC3T3-E1 cells. These results indicated that G_sα may be involved in regulation of physiological function, particularly in biological biomineralization.     

The complete amino-acid sequence of the rieske FeS-precursor protein from spinach chloroplasts deduced from cDNA analysis

Summary SummarySeveral cDNA clones encoding the entire Rieske FeS-precursor protein of the chloroplast cytochrome b ₆ f-complex have been isolated by high density plaque immunoscreening of a phage lambda gt11 cDNA expression library, made from poly A⁺-RNA of spinach seedlings. The identity of the cDNAs has been confirmed by N-terminal amino acid sequencing of the purified protein. The nucleotide sequence indicates a protein of 247 amino acid residues including a putative transit sequence of 68 amino acids corresponding to molecular masses of 26.3 kDa (precursor) and 18.8 kDa (mature protein; 179 amino acid residues). Alignteins of the sequence with sequences from Rieske FeS-proteins of respiratory electron transport chains, two of bacterial and three of mitochondrial origin, shows little sequence homology, but remarkable similarity in secondary structure including a putative N-terminal transmembrane segment of about 25 residues and the peptides CTHLGCV and CPCHGS in the C-terminal region of the protein that are involved in the binding of the Fe₂S₂-cluster.     

Bifunctional structure of two adenylyl cyclases from the myxobacterium Stigmatella aurantiaca

Two adenylyl cyclase genes (cyaA and cyaB) from the myxobacterium Stigmatella aurantiaca were cloned by complementation of Escherichia coli mutants defective in the cya gene. cyaA codes for a protein of 424 amino acid residues (AC1), while cyaB encodes a protein of 352 residues (AC2). Both cyclases are sensitive to adenosine: cAMP production was strongly inhibited in E coli cells and cell extracts expressing these genes. AC1 comprises a hydrophobic domain of six transmembrane helices coupled to a cytoplasmic catalytic domain endowed with adenylyl cyclase activity. A 17 amino acid residue sequence, which is a signature of G-protein coupled receptors, as well as of slime mold Dictyostelium discoideum cyclic AMP receptors, was found in the membrane domain. AC2 displays features also indicating that it is a bifunctional enzyme. The domain located upstream from the catalytic adenylyl cyclase domain shows strong similarity to receiver modules of response regulators of two-component bacterial signaling systems. In vitro mutagenesis of conserved aspartate residues in this domain was shown to interfere with cAMP synthesis.     

Sequence, expression and localization of the immunity protein for colicin B

Summary Cells of Escherichia coli containing the cbi locus on plasmids are immune to colicin B which kills cells by dissipating the membrane potential through pore formation in the cytoplasmic membrane. The nucleotide sequence of the cbi region was determined. It contains an open reading frame for a polypeptide consisting of 175 amino acids. The amino acid sequence is homologous to the primary structure of the colicin A immunity protein. This, and the strong homology between the pore-forming domains of colicins A and B suggests a common evolutionary origin for both colicins. The immunity protein could be identified following strong overexpression of cbi. The electrophoretically determined molecular weight of 20 000 was close to the calculated molecular weight of 20 185. The protein contains four large hydrophobic regions. The immunity protein was localized in the membrane fraction and was mainly contained in the cytoplasmic membrane. It is proposed that the immunity protein inactivates the colicin in the cytoplasmic membrane.     

Cloning and heterologous expression of a novel arylmalonate decarboxylase gene from Alcaligenes bronchisepticus KU 1201

We have cloned and sequenced a DNA fragment that encodes the arylmalonate decarboxylase (AMDase) gene from Alcaligenes bronchisepticus KU 1201. The AMDase gene consists of an open reading frame of 720 nucleotides, which specifies a 240-amino-acid protein of relative molecular mass (M_r) 24734. The M_r deduced from the AMDase gene is in good agreement with that of the AMDase isolated from A. bronchisepticus. No TATA or TTGA sequence was observed within the cloned DNA fragment, but the fragment was expressed in Escherichia coli by the lac promoter of pUC19. The enzyme produced in E. coli has the same M_r and the same enzyme activity as the purified from A. bronchisepticus. Comparison of the DNA sequence and the deduced amino acid sequence of AMDase with available DNA and amino acid sequence data bases revealed that there are no significant sequence homologies.Correspondence to: Hiromichi Ohta     

Complete amino acid sequence of the FK506 and rapamycin binding protein, FKBP, isolated from calf thymus

FKBP, an 11.8 kD intracellular protein that binds the immunosuppressants FK506 (K _d=0.4 nM) and rapamycin (K _d=0.2 nM) with high affinity, was purified to homogeneity from calf thymus. The complete amino acid sequence has been determined by automated Edman degradation of the intact molecule and overlapping fragments generated by proteolytic and chemical cleavage. The analysis revealed a 107 amino acid peptide chain with the following sequence: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFVLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPNATLIFDVELLKLE. The molecular weight, calculated from the amino sequence to be 11,778 D, was confirmed by electrospray ionization mass spectrometry. Thus, naturally isolated bovine FKBP does not appear to have any residues modified by glycosylation, phosphorylation, or other post-translational derivatization processes. Bovine FKBP has only three amino acid residues that differ from human FKBP, whose sequence was elucidated by cloning and sequencing complementary DNA (Standaertet al., 1990). The protein has a substantial number of hydrophilic peptide segments with prevalent -strand type of chain fold. Understanding the biological function of FKBP and other members of the immunophilin class and their respective complexes with immunosuppressive drugs may provide insights into cytoplasmic signalling mechanisms, protein folding and translocation, and other cellular processes.     

Characteristics of a novel secreted zinc-dependent endopeptidase of <Emphasis Type="Italic">Bacillus intermedius</Emphasis>

A novel zinc-dependent metalloendopeptidase of Bacillus intermedius (MprBi) was purified from the culture medium of a recombinant strain of Bacillus subtilis. The amino acid sequence of the homogeneous protein was determined using MALDI-TOF mass spectrometry. The sequence of the first ten residues from the N-terminus of the mature protein is ASTGSQKVTV. Physicochemical properties of the enzyme and its substrate specificity have been studied. The molecular weight of the metalloproteinase constitutes 19 kDa, the K _m and k _cat values are 0.06 mM and 1210 sec⁻¹, respectively, and the pI value is 5.4. The effect of different inhibitors and metal ions on the enzyme activity has been studied. Based on the analysis of the amino acid sequence of the active site motif and the Met-turn together with the enzyme characteristics, the novel bacterial metalloproteinase MprBi is identified as a metzincin clan adamalysin/reprolysin-like metalloprotease.     

The molecular organization of the lysostaphin gene and its sequences repeated in tandem

Summary The gene encoding lysostaphin of Staphylococcus staphylolyticus was cloned in Escherichia coli and its DNA sequence was determined. The complete coding region comprises 1440 base pairs corresponding to a precursor of 480 amino acids (molecular weight 51669). It was shown by NH₂-terminal amino acid sequence analysis of the purified extracellular lysostaphin from S. staphylolyticus that the mature lysostaphin consists of 246 amino acid residues (molecular weight 26926). Polyacrylamide gel electrophoresis revealed a similar molecular weight for the most active form. By computer analysis the secondary protein structure was predicted. It revealed three distinct regions in the precursor protein: a typical signal peptide (ca. 38 aa), a hydrophilic and highly ordered protein domain with 14 repetitive sequences (296 aa) and the hydrophobic mature lysostaphin. The lysostaphin precursor protein appears to be organized as a preprolysostaphin.Abbreviations aa amino acid(s)     

Beta-35 is a transferrin-derived inhibitor of angiogenesis and tumor growth

An angiogenesis inhibitor named Beta-35 has been identified and purified from the conditioned medium of mouse pancreatic β cells tumor cells. Beta-35 has a molecular weight of 35 kDa and inhibits DNA synthesis of bovine capillary endothelial cells at a half-maximal concentration of approximately 5 nM. It shows anti-angiogenic activity in the chick embryo chorioallantoic membrane at a dose of about 1 μg/embryo. Amino acid microsequencing and mass spectrometric analysis of the purified protein demonstrate that Beta-35 contains the first 314 residues of the N-terminal sequence of bovine transferrin. We have cloned and expressed this protein in Escherichia coli using the corresponding gene segment of Beta-35 contained in the cDNA of human transferrin. The recombinant protein of Beta-35 shows significant anti-tumor activity at a dose of 5 mg/kg/day against human pancreatic cancer or melanoma implanted subcutaneously in SCID mice.     

Purification and Amino Acid Sequence of MP-III 4R D49 Phospholipase A2 from Bothrops pirajai Snake Venom, a Toxin with Moderate PLA2 and Anticoagulant Activities and High Myotoxic Activity

MP-III 4R PLA₂ was purified from the venom of Bothrops pirajai venom (Bahia's jararacussu) after three chromatographic steps which started with RP-HPLC. The complete amino acid sequence of MP-III 4R PLA₂ from Bothrops pirajai was determined by amino acid sequencing of reduced and carboxymethylated MP-III 4R and the isolated peptides from clostripain and protease V8 digestion. MP-III 4R is a D49 PLA₂ with 121 amino acid residues and has a molecular weight estimated at 13,800 Da, with 14 half-cysteines. This protein showed moderate PLA₂ and anticoagulant activity. This PLA₂ does not have a high degree of homology with other bothropic PLA₂-like myotoxins (~75%) and nonbothropic myotoxins (~60%). MP-III 4R is a new PLA₂, which was isolated using exclusively analytical and preparative HPLC methods. Based on the N-terminal sequence and biological activities, MP-III 4R was identified as similar to piratoxin-III (PrTX-III), which was isolated by conventional chromatography based on molecular exclusion ion exchange chromatography. Clinical manifestations indicate that at the site of toxin injection, there may be pain of variable intensity, because animals continue to lick the limb. No clinical sign indicating general toxicity was noticed. Myotoxicity was observed in gastrocnemius muscle cells after exposure to MP-III 4R, with a high frequency (70%) of affected muscle fibers.     

Purification, Biological Activities, and Molecular Cloning of a Novel Mannose-Binding Lectin from Bulbs of Zephyranthes candida Herb （Amaryllidaceae）

A novel mannose-bindlng aggiutinln was purified from bulbs of Zephyranthes candida Herb by extraction, precipitation with 80% （NH4）2SO4, and ion-exchange chromatography on DEAE-Sepharose followed by gel flitration on Sephscryl S-100. The purified Z. candida agglutlnln （ZCA） migrated as a single band of 12 kDa on sodium dodecyi suifate-poiyecryiamide gel electrophoresis under reducing and non-reducing conditions. The apparent molecular mass of the iectln, as datermlned by gel filtration chromatography, was 48 kDa. The results Indicated that ZCA was composed of four Identical subunlts of 12 kDa each （homotetramerlc nature）. The ZCA agglutlhated rabbit erythrocytes, Escherichla coil and Saccharomyces cerevislae ceils at concentrations of 0.95, 1.90, and 31.30 μg/mL, respectively. Bloassays Indicated that ZCA has a significant effect on wheat aphid survival. Mortality after 7 d was 〉 90% at 0.26%. A degenerate primer was designed In accordance with the N-terminal partial sequence of purified ZCA. The full-length cDNA was cloned by 3＇- and 5＇-rapid amplification of cDNA ends. The full-length cDNA had 661 bp and the sequence encoded an open reading frame of 168 amino acids. The mature protein of ZCA Includes 109 amino acid residues and the molecular weight of the protein was 12.1 kDa. The result show that the zca gene encodes a protein precursor with a signal peptlde, a mature protein, and a C-terminal cleavage amino acids sequence. Molecular modeling of ZCA Indicated that Its three-dimensional atructure strongly resembies that of the snowdrop aggiutinin. Blocks＇ analysis revealed that the deduced amino acid sequence of ZCA has three functional domains specific for agglutination and three carbohydrate binding boxes （QDNY）.     

Low expression of lipid-linked oligosaccharide due to a functionally altered Dol-P-Man synthase reduces protein glycosylation in cAMP-dependent protein kinase deficient Chinese hamster ovary cells

Chinese hamster ovary cells express a wide variety of glycoproteins with M_r ranging from 15,000 to 200,000 dalton and higher. Glycosylation of these proteins was much less in cAMP-dependent protein kinase (PKA)-deficient mutants which expressed either (i) a defective C-subunit with altered substrate specificity and having no detectable type II kinase (mutant 10215); or (ii) an altered RI subunit and having no detectable type II kinase (mutant 10248); or (iii) exhibited the lowest level of total kinase with no detectable type I kinase but having a small amount of type II kinase (mutant 10260). Addition of 8Br-cAMP enhanced protein glycosylation index in wild type cells 10001 by 120% but only 7 to 23% in the mutant cells. The rate of lipid-linked oligosaccharide (LLO) biosynthesis was linear for 1 h in all cell types, but the total amount of LLO expressed was much less in PKA-deficient mutants. Pulse-chase experiments indicated that the t_1/2 for LLO turnover was also twice as high in PKA-deficient cells as in the wild type. Size exclusion chromatography of the mild-acid released oligosaccharide confirmed that both wild type and the mutant cells synthesized Glc₃Man₉GlcNAc₂-PP-Dol as the most predominating species with no accumulation of Man₅GlcNAc₂-PP-Dol in the mutants. Kinetic studies exhibited a reduced mannosylphosphodolichol synthase (DPMS) activity in mutant cells with a K_m for GDP-mannose 160 to 400% higher than that of the wild type. In addition, the k_cat for DPMS was also reduced 2 to 4-fold in these mutant cells. Exogenously added Dol-P failed to rescue the k_cat for DPMS in CHO cell mutants; however, in vitro protein phosphorylation with a cAMP-dependent protein kinase restored their kinetic activity to the level of the wild type. Published in 2004.     

The Helicobacter felis ftsH gene encoding an ATP-dependent metalloprotease can replace the Escherichia coli homologue for growth and phage λ lysogenization

Cloning and sequencing of an approximately 6.0-kb chromosomal DNA fragment from Helicobacter felis revealed five complete open reading frames. The deduced amino acid sequence of one ORF exhibited sequence similarity to the FtsH protein, an ATP-dependent metalloprotease, from various bacterial species. The encoded protein consists of 638 amino acid residues with a molecular mass of 70.2 kDa. The hydropathy profile of the FtsH protein predicted two N-terminal transmembrane regions that were confirmed experimentally. Insertion of ftsH into a new versatile expression vector resulted in overexpression of FtsH protein in Escherichia coli. In addition, the E. coli ftsH gene could be replaced by the H. felis homologue to allow reduced growth and tenfold increased lysogenization by temperate phage λ. Received: 6 November 1997 / Accepted: 22 January 1998     

Overexpression of a small GTP-binding protein Ran1 in Arabidopsis leads to promoted elongation growth and enhanced disease resistance against P. syringae DC3000

Plants resist infection through an innate immune response, which is usually associated with slowing of growth. The molecular mechanisms underlying the trade-off between plant growth and defense remain unclear. The present study reveals that growth/defense trade-offs mediated by gibberellin (GA) and salicylic acid (SA) signaling pathways are uncoupled during constitutive overexpression of transgenic AtRAN1 and AtRAN1_Q72L (active, GTP-locked form) Arabidopsis plants. It is well known that the small GTP-binding protein Ran (a Ras-related nuclear protein) functions in the nucleus–cytoplasmic transport of proteins. Although there is considerable evidence indicating that nuclear–cytoplasmic partitioning of specific proteins can participate in hormone signaling, the role of Ran-dependent nuclear transport in hormone signaling is not yet fully understood. In this report, we used a combination of genetic and molecular methods to reveal whether AtRAN1 is involved in both GA and SA signaling pathways. Constitutively overexpressed AtRAN1 promoted both elongation growth and the disease resistance response, whereas overexpression of AtRAN1_Q72L in the atran2atran3 double mutant background clearly inhibited elongation growth and the defense response. Furthermore, we found that AtRAN1 coordinated plant growth and defense by promoting the stability of the DELLA protein RGA in the nucleus and by modulating NPR1 nuclear localization. Interestingly, genetically modified rice (Oryza sativa) overexpressing AtRAN1 exhibited increased plant height and yield per plant. Altogether, the ability to achieve growth/defense trade-offs through AtRAN1 overexpression provides an approach to maximizing crop yield to meet rising global food demands.     

LKB1 Deficiency in Tie2-Cre-expressing Cells Impairs Ischemia-induced Angiogenesis

LKB1 is a tumor suppressor protein whose loss leads to HIF1α-mediated activation of a proangiogenic program in intestinal polyps. LKB1 is also protein kinase regulator of AMP-activated protein kinase (AMPK) signaling, which is essential for endothelial cell responses to tissue ischemia. To discern whether LKB1 signaling is either pro- or antiangiogenic, we investigated ischemia-induced revascularization in mice that were deficient for LKB1 in Tie2-Cre-expressing cells. Whereas homozygous deletion of LKB1 led to embryonic lethality, heterozygous LKB1-knock-out (KO) (Lkb1^flox/+;Tie2^Tg/+) mice were viable. Unchallenged heterozygous LKB1-KO mice displayed normal capillary density, but the revascularization of hind limb following ischemic surgery was significantly impaired as evaluated by laser Doppler flow and capillary density measurements. Reduction of LKB1 in cultured endothelial cells, using either small interfering RNA or an adenovirus expressing nonfunctional kinase-dead LKB1 protein, attenuated endothelial proliferation, migration, and differentiation into network structures on Matrigel that was accompanied by diminished AMPK phosphorylation at Thr-172. Conversely, adenovirus-mediated LKB1 overexpression (Ad-LKB1) augmented network structure formation, and this was associated with elevated AMPK phosphorylation. The augmented differentiation of endothelial cells into network structures induced by Ad-LKB1 was abrogated by the co-transduction of a dominant negative mutant of AMPK. These observations suggest that the LKB1-AMPK signaling axis in endothelial cells is a positive regulator of the revascularization response to tissue ischemia.     

The primary structure of a cDNA for PsaN,encoding an extrinsic lumenal polypeptide of barley photosystem I

A cDNA clone encoding a 15.501 Da photosystem I (PSI) subunit of barley was isolated using an oligonucleotide based on the NH₂-terminal amino acid sequence of the isolated protein. The polypeptide, which migrates with an apparent molecular mass of 9.5 kDa on denaturing SDS-PAGE, has been designated PSI-N, and the corresponding gene is PsaN. Analysis of the deduced protein sequence indicates a mature protein of 85 amino acid residues and a molecular mass of 9818 Da. PSI-N is a hydrophilic, extrinsic protein with no predicted membrane-spanning regions. The transit peptide of 60 residues (5683 Da) contains a predicted hydrophobic -helix, suggesting that the protein is routed into the thylakoid lumen. Thus, PSI-N is the second known lumenal protein component associated with PSI, together with PSI-F.     

Synthesis of adhesive protein from the vitellaria of the liver flukeFasciola hepatica

Summary The polynonapeptide (Gly-Gly-Gly-Tyr-Gly-Gly-Tyr-Gly-Lys)_n, which is a precursor sequence of adhesive protein from the vitellaria of the liver flukeFasciola hepatica has been synthesized by the fragment coupling, followed by polycondensation, and by cleavage of the protecting groups by hydrogen bromide. The synthetic adhesive protein was estimated to have the molecular weight of 10,100 (12 repeating units as nonapeptide) and was found to have satisfactory amino acid compositions. The Tyr residues of the synthesized precursor polynonapeptides can be converted to the Dopa residues by tyrosinase, giving the synthetic adhesive protein of the liver fluke.