Molecular cloning and expression of a UDP-N-acetylglucosamine (GlcNAc):hydroxyproline polypeptide GlcNAc-transferase that modifies Skp1 in the cytoplasm of dictyostelium

Skp1 is a ubiquitous eukaryotic protein found in several cytoplasmic and nuclear protein complexes, including the SCF-type E3 ubiquitin ligase. In Dictyostelium, Skp1 is hydroxylated at proline 143, which is then modified by a pentasaccharide chain. The enzyme activity that attaches the first sugar, GlcNAc, was previously shown to copurify with the GnT51 polypeptide whose gene has now been cloned using a proteomics approach based on a quadrupole/time-of-flight hybrid mass spectrometer. When expressed in Escherichia coli, recombinant GnT51 exhibits UDP-GlcNAc:hydroxyproline Skp1 GlcNAc-transferase activity. Based on amino acid sequence alignments, GnT51 defines a new family of microbial polypeptide glycosyltransferases that appear to be distantly related to the catalytic domain of mucin-type UDP-GalNAc:Ser/Thr polypeptide alpha-GalNAc-transferases expressed in the Golgi compartment of animal cells. This relationship is supported by the effects of site-directed mutagenesis of GnT51 amino acids associated with its predicted DXD-like motif, DAH. In contrast, GnT51 lacks the N-terminal signal anchor sequence present in the Golgi enzymes, consistent with the cytoplasmic localization of the Skp1 acceptor substrate and the biochemical properties of the enzyme. The first glycosylation step of Dictyostelium Skp1 is concluded to be mechanistically similar to that of animal mucin type O-linked glycosylation, except that it occurs in the cytoplasm rather than the Golgi compartment of the cell.     

Molecular characterization of a novel UDP-galactose:fucoside alpha3-galactosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium

Skp1 is a nucleocytoplasmic protein that is post-translationally modified by a pentasaccharide, Gal alpha1,Gal alpha1,3Fuc alpha1,2Gal-beta1,3GlcNAc alpha1O-, at a 4-hydroxylated derivative of Pro-143 in the amebazoan Dictyostelium discoideum. An enzymatic activity that catalyzes formation of the Gal alpha1,3Fuc linkage by transfer of Gal from UDP-alphaGal to Fuc alpha1,2Gal beta1,3GlcNAc alpha1O-benzyl, or the corresponding glycoform of Skp1, was described previously in cytosolic extracts of Dictyostelium. A protein GT78 associated with this activity has been purified to chromatographic homogeneity. In-gel tryptic digestion followed by nano-liquid chromatography-mass spectrometry on a quadrupole time-of-flight geometry instrument with data-dependent tandem mass spectrometry acquisition yielded a number of peptide fragmentation spectra, nine of which were manually de novo sequenced and found to map onto a predicted 3-exon gene of unknown function on chromosome 4. GT78 is predicted to comprise 648 amino acids with an N-terminal glycosyltransferase and a C-terminal beta-propeller domain. Overexpression of GT78 with a His6-tag resulted in a 120-fold increase in GalT-activity in cytosolic extracts, and purified His6-GT78 exhibited alpha3GalT-activity toward a synthetic acceptor substrate. Expression of the truncated N-terminal region confirmed the predicted catalytic activity of this domain. Disruption of the GT78 gene led to a loss of enzyme activity in extracts and accumulation of the non-galactosylated isoform of Skp1 in cells. GT78 therefore represents the Skp1 alpha3GalT, and its mechanism conforms to the sequential model of Skp1 glycosylation in the cytoplasm shown for earlier enzymes in the pathway. Informatics studies suggest that related catalytic domains are expressed in the Golgi or cytoplasm of plants, other protozoans, and animals.     

Reduction of collagen production in keloid fibroblast cultures by ethyl-3,4-dihydroxybenzoate. Inhibition of prolyl hydroxylase activity as a mechanism of action

Excessive accumulation of collagen is the hallmark of several clinical conditions characterized by tissue fibrosis. Previously, 3,4-dihydroxybenzoic acid, a structural analog of alpha-ketoglutarate and ascorbate, has been shown to inhibit the activity of purified prolyl 4-hydroxylase, the enzyme catalyzing the synthesis of 4-hydroxyproline during intracellular biosynthesis of procollagen. In this study a hydrophobic modification, an ethyl ester, of 3,4-dihydroxybenzoic acid was tested for its effects on collagen synthesis and prolyl hydroxylase activity in human skin fibroblast cultures. The results indicated that 0.4 mM ethyl-3,4-dihydroxybenzoate markedly inhibited the synthesis of 4-hydroxyproline in normal cell cultures apparently as a result of reduced prolyl 4-hydroxylase activity, and the synthesis and secretion of both type I and type III procollagens were markedly reduced. Control experiments indicated that the test compound did not affect the viability, proliferation, or plating efficiency of the cells, and it had little, if any, effect on the synthesis of noncollagenous proteins. Furthermore, determinations of type I and type III procollagen mRNA steady-state levels by slot-blot hybridizations suggested that the inhibition of procollagen production did not occur on the pretranslational level. Thus, ethyl-3,4-dihydroxybenzoate selectively reduced procollagen production in fibroblast cultures by inhibiting the post-translational synthesis of 4-hydroxyproline. Similar inhibition was also observed in keloid fibroblast cultures, demonstrating the potential applicability of ethyl-3,4-dihydroxybenzoate, or other structural alpha-ketoglutarate or ascorbate analogs, for treatment of fibrotic diseases.     

Initiation of mucin-type O-glycosylation in dictyostelium is homologous to the corresponding step in animals and is important for spore coat function

Like animal cells, many unicellular eukaryotes modify mucin-like domains of secretory proteins with multiple O-linked glycans. Unlike animal mucin-type glycans, those of some microbial eukaryotes are initiated by alpha-linked GlcNAc rather than alpha-GalNAc. Based on sequence similarity to a recently cloned soluble polypeptide hydroxyproline GlcNAc-transferase that modifies Skp1 in the cytoplasm of the social ameba Dictyostelium, we have identified an enzyme, polypeptide alpha-N-acetylglucosaminyltransferase (pp alpha-GlcNAc-T2), that attaches GlcNAc to numerous secretory proteins in this organism. Unlike the Skp1 GlcNAc-transferase, pp alpha-GlcNAc-T2 is predicted to be a type 2 transmembrane protein. A highly purified, soluble, recombinant fragment of pp alpha-GlcNAc-T2 efficiently transfers GlcNAc from UDP-GlcNAc to synthetic peptides corresponding to mucin-like domains in two proteins that traverse the secretory pathway. pp alpha-GlcNAc-T2 is required for addition of GlcNAc to peptides in cell extracts and to the proteins in vivo. Mass spectrometry and Edman degradation analyses show that pp alpha-GlcNAc-T2 attaches GlcNAc in alpha-linkage to the Thr residues of all the synthetic mucin repeats. pp alpha-GlcNAc-T2 is encoded by the previously described modB locus defined by chemical mutagenesis, based on sequence analysis and complementation studies. This finding establishes that the many phenotypes of modB mutants, including a permeability defect in the spore coat, can now be ascribed to defects in mucin-type O-glycosylation. A comparison of the sequences of pp alpha-GlcNAc-T2 and the animal pp alpha-GalNAc-transferases reveals an ancient common ancestry indicating that, despite the different N-acetylhexosamines involved, the enzymes share a common mechanism of action.     

An endoplasmic reticulum transmembrane prolyl 4-hydroxylase is induced by hypoxia and acts on hypoxia-inducible factor alpha

Prolyl 4-hydroxylases (P4Hs) act on collagens (C-P4Hs) and the oxygen-dependent degradation domains (ODDDs) of hypoxia-inducible factor alpha subunits (HIF-P4Hs) leading to degradation of the latter. We report data on a human P4H possessing a transmembrane domain (P4H-TM). Its gene is also found in zebrafish but not in flies and nematodes. Its sequence more closely resembles those of the C-P4Hs than the HIF-P4Hs, but it lacks the peptide substrate-binding domain of the C-P4Hs. P4H-TM levels in cultured cells are increased by hypoxia, and P4H-TM is N-glycosylated and is located in endoplasmic reticulum membranes with its catalytic site inside the lumen, a location differing from those of the HIF-P4Hs. Despite this, P4H-TM overexpression in cultured neuroblastoma cells reduced HIF-alpha ODDD reporter construct levels, and its small interfering RNA increased HIF-1alpha protein level, in the same way as those of HIF-P4Hs. Furthermore, recombinant P4H-TM hydroxylated the two critical prolines in HIF-1alpha ODDD in vitro, with a preference for the C-terminal proline, whereas it did not hydroxylate any prolines in recombinant type I procollagen chains.     

Prolyl hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium

O₂ regulates multicellular development of the social amoeba Dictyostelium, suggesting it may serve as an important cue in its native soil environment. Dictyostelium expresses an HIFα-type prolyl 4-hydroxylase (P4H1) whose levels affect the O₂-threshold for culmination implicating it as a direct O₂-sensor, as in animals. But Dictyostelium lacks HIFα, a mediator of animal prolyl 4-hydroxylase signaling, and P4H1 can hydroxylate Pro143 of Skp1, a subunit of E3^SCFubiquitin-ligases. Skp1 hydroxyproline then becomes the target of five sequential glycosyltransferase reactions that modulate the O₂-signal. Here we show that genetically induced changes in Skp1 levels also affect the O₂-threshold, in opposite direction to that of the modification enzymes suggesting that the latter reduce Skp1 activity. Consistent with this, overexpressed Skp1 is poorly hydroxylated and Skp1 is the only P4H1 substrate detectable in extracts. Effects of Pro143 mutations, and of combinations of Skp1 and enzyme level perturbations, are consistent with pathway modulation of Skp1 activity. However, some effects were not mirrored by changes in modification of the bulk Skp1 pool, implicating a Skp1 subpopulation and possibly additional unknown factors. Altered Skp1 levels also affected other developmental transitions in a modification-dependent fashion. Whereas hydroxylation of animal HIFα results in its polyubiquitination and proteasomal degradation, Dictyostelium Skp1 levels were little affected by its modification status. These data indicate that Skp1 and possibly E3^SCFubiquitin-ligase activity modulate O₂-dependent culmination and other developmental processes, and at least partially mediate the action of the hydroxylation/glycosylation pathway in O₂-sensing.     

Complex glycosylation of Skp1 in Dictyostelium: implications for the modification of other eukaryotic cytoplasmic and nuclear proteins

Recently, complex O-glycosylation of the cytoplasmic/nuclear protein Skp1 has been characterized in the eukaryotic microorganism Dictyostelium. Skp1's glycosylation is mediated by the sequential action of a prolyl hydroxylase and five conventional sugar nucleotide-dependent glycosyltransferase activities that reside in the cytoplasm rather than the secretory compartment. The Skp1-HyPro GlcNAcTransferase, which adds the first sugar, appears to be related to a lineage of enzymes that originated in the prokaryotic cytoplasm and initiates mucin-type O-linked glycosylation in the lumen of the eukaryotic Golgi apparatus. GlcNAc is extended by a bifunctional glycosyltransferase that mediates the ordered addition of beta1,3-linked Gal and alpha1,2-linked Fuc. The architecture of this enzyme resembles that of certain two-domain prokaryotic glycosyltransferases. The catalytic domains are related to those of a large family of prokaryotic and eukaryotic, cytoplasmic, membrane-bound, inverting glycosyltransferases that modify glycolipids and polysaccharides prior to their translocation across membranes toward the secretory pathway or the cell exterior. The existence of these enzymes in the eukaryotic cytoplasm away from membranes and their ability to modify protein acceptors expose a new set of cytoplasmic and nuclear proteins to potential prolyl hydroxylation and complex O-linked glycosylation.     

High-level production of human collagen prolyl 4-hydroxylase in Escherichia coli.

The collagen prolyl 4-hydroxylases (C-P4Hs), enzymes residing within the lumen of the endoplasmic reticulum, play a central role in the synthesis of all collagens. The vertebrate enzymes are alpha(2)beta(2) tetramers in which the two catalytic sites are located in the alpha subunits, and protein disulfide isomerase serves as the beta subunit. All attempts to assemble an active C-P4H tetramer from its subunits in in vitro cell-free systems have been unsuccessful, but assembly of a recombinant enzyme has been reported in several cell types by coexpression of the two types of subunit. An active type I C-P4H tetramer was obtained here by periplasmic expression in Escherichia coli strains BL21 and RB791. Further optimization for production by stepwise regulated coexpression of its subunits in the cytoplasm of a thioredoxin reductase and glutathione reductase mutant E. coli strain resulted in large amounts of human type I C-P4H tetramer. The specific activity of the C-P4H tetramer purified from the cytoplasmic expression was within the range of values reported for human type I C-P4H isolated as a nonrecombinant enzyme or produced in the endoplasmic reticulum of insect cells, but the expression level, about 25 mg/l in a fermenter, is about 5-10 times that obtained in insect cells. The enzyme expressed in E. coli differed from those present in vivo and those produced in other hosts in that it lacked the N glycosylation of its alpha subunits, which may be advantageous in crystallization experiments.     

Activation and partial characterization of prolyl hydroxylase from regenerating limbs of the adult newt Notophthalmus viridescens

Prolyl hydroxylase activity extracted from regenerating newt (Notophthalmus viridescens) limb tissues can be increased by brief preincubation with cofactors (ascorbate, alpha-ketoglutarate, Fe2+ and O2) prior to assay with [3H]proline-labeled collagen substrate. Newt prolyl hydroxylase is optimally active at 30 degrees C, but loses activity rapidly at 37 degrees C. The presence of cofactors or substrate decreases enzyme heat lability. Detergents (Triton X-100 and octyl glucoside) do not aid enzyme extraction and inhibit enzyme activity. Activity solubilized during homogenization without detergent remains soluble following high speed centrifugations. Comparative studies are reported for enzyme extracted from chick embryo tissues.     

Crystal structure and expression patterns of prolyl 4-hydroxylases from Phytophthora capsici

Prolyl 4-hydroxylases (P4Hs) are members of the Fe²⁺ and 2-oxoglutarate- dependent oxygenases family, which play central roles in the collagen stabilization, hypoxia sensing, and translational regulation in eukaryotes. Thus far, nothing is known about the role of P4Hs in development and pathogenesis in oomycetes. Here we show that the Phytophthora capsici genome contains five putative prolyl 4-hydroxylases. In mycelia, all P4Hs were downregulated in response to hypoxia, but the expression of PcP4H1 was most affected. Strikingly, Pc4H1 was upregulated more than 110 fold at the onset of infection, and Pc4H5 was upregulated seven fold, while the expression of other P4H's were unchanged. Similar to well-characterized P4H proteins, the crystallographic structure of PcP4H1 contains a highly conserved double-stranded β-helix core fold and catalytic residues. However, the binding affinity of 2-oxoglutarate to PcP4H1 is very low. The extended C-terminal α-helix bundle and longer β2-β3 disordered substrate binding loop may help in confirming the peptide target of this enzyme.