After translation, many newly formed proteins undergo further covalent modifications that alter their functional properties and that are essentially irreversible under physiological conditions in the body. These modifications include the vitamin K-dependent attachment of carboxyl groups to glutamate residues and the conversion of a lysine residue in eIF5A to hypusine, and the conversion of a histidine residue in EEF to diphthamide.
View original pathway at Reactome.
Abdel-Fattah W, Scheidt V, Uthman S, Stark MJ, Schaffrath R.; ''Insights into diphthamide, key diphtheria toxin effector.''; PubMedEurope PMCScholia
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Stenina O, Pudota BN, McNally BA, Hommema EL, Berkner KL.; ''Tethered processivity of the vitamin K-dependent carboxylase: factor IX is efficiently modified in a mechanism which distinguishes Gla's from Glu's and which accounts for comprehensive carboxylation in vivo.''; PubMedEurope PMCScholia
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Manfioletti G, Brancolini C, Avanzi G, Schneider C.; ''The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade.''; PubMedEurope PMCScholia
Lin Z, Su X, Chen W, Ci B, Zhang S, Lin H.; ''Dph7 catalyzes a previously unknown demethylation step in diphthamide biosynthesis.''; PubMedEurope PMCScholia
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Mariappan M, Preusser-Kunze A, Balleininger M, Eiselt N, Schmidt B, Gande SL, Wenzel D, Dierks T, von Figura K.; ''Expression, localization, structural, and functional characterization of pFGE, the paralog of the Calpha-formylglycine-generating enzyme.''; PubMedEurope PMCScholia
Poser JW, Esch FS, Ling NC, Price PA.; ''Isolation and sequence of the vitamin K-dependent protein from human bone. Undercarboxylation of the first glutamic acid residue.''; PubMedEurope PMCScholia
Ferron M, Lacombe J, Germain A, Oury F, Karsenty G.; ''GGCX and VKORC1 inhibit osteocalcin endocrine functions.''; PubMedEurope PMCScholia
Wei H, Bera TK, Wayne AS, Xiang L, Colantonio S, Chertov O, Pastan I.; ''A modified form of diphthamide causes immunotoxin resistance in a lymphoma cell line with a deletion of the WDR85 gene.''; PubMedEurope PMCScholia
Yang J, Kulkarni K, Manolaridis I, Zhang Z, Dodd RB, Mas-Droux C, Barford D.; ''Mechanism of isoprenylcysteine carboxyl methylation from the crystal structure of the integral membrane methyltransferase ICMT.''; PubMedEurope PMCScholia
Ware J, Diuguid DL, Liebman HA, Rabiet MJ, Kasper CK, Furie BC, Furie B, Stafford DW.; ''Factor IX San Dimas. Substitution of glutamine for Arg-4 in the propeptide leads to incomplete gamma-carboxylation and altered phospholipid binding properties.''; PubMedEurope PMCScholia
Hauschka PV, Lian JB, Cole DE, Gundberg CM.; ''Osteocalcin and matrix Gla protein: vitamin K-dependent proteins in bone.''; PubMedEurope PMCScholia
Landgrebe J, Dierks T, Schmidt B, von Figura K.; ''The human SUMF1 gene, required for posttranslational sulfatase modification, defines a new gene family which is conserved from pro- to eukaryotes.''; PubMedEurope PMCScholia
Zhang Y, Zhu X, Torelli AT, Lee M, Dzikovski B, Koralewski RM, Wang E, Freed J, Krebs C, Ealick SE, Lin H.; ''Diphthamide biosynthesis requires an organic radical generated by an iron-sulphur enzyme.''; PubMedEurope PMCScholia
Ouyang Yb, Lane WS, Moore KL.; ''Tyrosylprotein sulfotransferase: purification and molecular cloning of an enzyme that catalyzes tyrosine O-sulfation, a common posttranslational modification of eukaryotic proteins.''; PubMedEurope PMCScholia
DiScipio RG, Davie EW.; ''Characterization of protein S, a gamma-carboxyglutamic acid containing protein from bovine and human plasma.''; PubMedEurope PMCScholia
Dierks T, Schmidt B, Borissenko LV, Peng J, Preusser A, Mariappan M, von Figura K.; ''Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme.''; PubMedEurope PMCScholia
Diver MM, Long SB.; ''Mutational analysis of the integral membrane methyltransferase isoprenylcysteine carboxyl methyltransferase (ICMT) reveals potential substrate binding sites.''; PubMedEurope PMCScholia
Kang KR, Kim YS, Wolff EC, Park MH.; ''Specificity of the deoxyhypusine hydroxylase-eukaryotic translation initiation factor (eIF5A) interaction: identification of amino acid residues of the enzyme required for binding of its substrate, deoxyhypusine-containing eIF5A.''; PubMedEurope PMCScholia
Cosma MP, Pepe S, Annunziata I, Newbold RF, Grompe M, Parenti G, Ballabio A.; ''The multiple sulfatase deficiency gene encodes an essential and limiting factor for the activity of sulfatases.''; PubMedEurope PMCScholia
Dong M, Su X, Dzikovski B, Dando EE, Zhu X, Du J, Freed JH, Lin H.; ''Dph3 is an electron donor for Dph1-Dph2 in the first step of eukaryotic diphthamide biosynthesis.''; PubMedEurope PMCScholia
Su X, Lin Z, Chen W, Jiang H, Zhang S, Lin H.; ''Chemogenomic approach identified yeast YLR143W as diphthamide synthetase.''; PubMedEurope PMCScholia
Delpierrre G, Vertommen D, Communi D, Rider MH, Van Schaftingen E.; ''Identification of fructosamine residues deglycated by fructosamine-3-kinase in human hemoglobin.''; PubMedEurope PMCScholia
Foster DC, Sprecher CA, Holly RD, Gambee JE, Walker KM, Kumar AA.; ''Endoproteolytic processing of the dibasic cleavage site in the human protein C precursor in transfected mammalian cells: effects of sequence alterations on efficiency of cleavage.''; PubMedEurope PMCScholia
Moehring JM, Moehring TJ.; ''The post-translational trimethylation of diphthamide studied in vitro.''; PubMedEurope PMCScholia
Murphy JR.; ''Mechanism of diphtheria toxin catalytic domain delivery to the eukaryotic cell cytosol and the cellular factors that directly participate in the process.''; PubMedEurope PMCScholia
Wright LP, Court H, Mor A, Ahearn IM, Casey PJ, Philips MR.; ''Topology of mammalian isoprenylcysteine carboxyl methyltransferase determined in live cells with a fluorescent probe.''; PubMedEurope PMCScholia
Shearer MJ, Fu X, Booth SL.; ''Vitamin K nutrition, metabolism, and requirements: current concepts and future research.''; PubMedEurope PMCScholia
Thim L, Bjoern S, Christensen M, Nicolaisen EM, Lund-Hansen T, Pedersen AH, Hedner U.; ''Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells.''; PubMedEurope PMCScholia
Collard F, Delpierre G, Stroobant V, Matthijs G, Van Schaftingen E.; ''A mammalian protein homologous to fructosamine-3-kinase is a ketosamine-3-kinase acting on psicosamines and ribulosamines but not on fructosamines.''; PubMedEurope PMCScholia
Degen SJ, Davie EW.; ''Nucleotide sequence of the gene for human prothrombin.''; PubMedEurope PMCScholia
von Figura K, Schmidt B, Selmer T, Dierks T.; ''A novel protein modification generating an aldehyde group in sulfatases: its role in catalysis and disease.''; PubMedEurope PMCScholia
Sjölinder M, Uhlmann J, Ponstingl H.; ''Characterisation of an evolutionary conserved protein interacting with the putative guanine nucleotide exchange factor DelGEF and modulating secretion.''; PubMedEurope PMCScholia
McMullen BA, Fujikawa K, Kisiel W.; ''The occurrence of beta-hydroxyaspartic acid in the vitamin K-dependent blood coagulation zymogens.''; PubMedEurope PMCScholia
Kim YS, Kang KR, Wolff EC, Bell JK, McPhie P, Park MH.; ''Deoxyhypusine hydroxylase is a Fe(II)-dependent, HEAT-repeat enzyme. Identification of amino acid residues critical for Fe(II) binding and catalysis [corrected].''; PubMedEurope PMCScholia
Danan LM, Yu Z, Hoffhines AJ, Moore KL, Leary JA.; ''Mass spectrometric kinetic analysis of human tyrosylprotein sulfotransferase-1 and -2.''; PubMedEurope PMCScholia
Van Ness BG, Howard JB, Bodley JW.; ''ADP-ribosylation of elongation factor 2 by diphtheria toxin. Isolation and properties of the novel ribosyl-amino acid and its hydrolysis products.''; PubMedEurope PMCScholia
Joe YA, Wolff EC, Park MH.; ''Cloning and expression of human deoxyhypusine synthase cDNA. Structure-function studies with the recombinant enzyme and mutant proteins.''; PubMedEurope PMCScholia
Shearer MJ, Newman P.; ''Recent trends in the metabolism and cell biology of vitamin K with special reference to vitamin K cycling and MK-4 biosynthesis.''; PubMedEurope PMCScholia
Walz DA, Hewett-Emmett D, Seegers WH.; ''Amino acid sequence of human prothrombin fragments 1 and 2.''; PubMedEurope PMCScholia
Leytus SP, Chung DW, Kisiel W, Kurachi K, Davie EW.; ''Characterization of a cDNA coding for human factor X.''; PubMedEurope PMCScholia
Mattheakis LC, Shen WH, Collier RJ.; ''DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae.''; PubMedEurope PMCScholia
Teramoto T, Fujikawa Y, Kawaguchi Y, Kurogi K, Soejima M, Adachi R, Nakanishi Y, Mishiro-Sato E, Liu MC, Sakakibara Y, Suiko M, Kimura M, Kakuta Y.; ''Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction.''; PubMedEurope PMCScholia
Liu S, Milne GT, Kuremsky JG, Fink GR, Leppla SH.; ''Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2.''; PubMedEurope PMCScholia
Hammed A, Matagrin B, Spohn G, Prouillac C, Benoit E, Lattard V.; ''VKORC1L1, an enzyme rescuing the vitamin K 2,3-epoxide reductase activity in some extrahepatic tissues during anticoagulation therapy.''; PubMedEurope PMCScholia
Park MH.; ''The post-translational synthesis of a polyamine-derived amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF5A).''; PubMedEurope PMCScholia
Hagen FS, Gray CL, O'Hara P, Grant FJ, Saari GC, Woodbury RG, Hart CE, Insley M, Kisiel W, Kurachi K.; ''Characterization of a cDNA coding for human factor VII.''; PubMedEurope PMCScholia
Liu S, Leppla SH.; ''Retroviral insertional mutagenesis identifies a small protein required for synthesis of diphthamide, the target of bacterial ADP-ribosylating toxins.''; PubMedEurope PMCScholia
Preusser-Kunze A, Mariappan M, Schmidt B, Gande SL, Mutenda K, Wenzel D, von Figura K, Dierks T.; ''Molecular characterization of the human Calpha-formylglycine-generating enzyme.''; PubMedEurope PMCScholia
Bär C, Zabel R, Liu S, Stark MJ, Schaffrath R.; ''A versatile partner of eukaryotic protein complexes that is involved in multiple biological processes: Kti11/Dph3.''; PubMedEurope PMCScholia
Park JH, Wolff EC, Folk JE, Park MH.; ''Reversal of the deoxyhypusine synthesis reaction. Generation of spermidine or homospermidine from deoxyhypusine by deoxyhypusine synthase.''; PubMedEurope PMCScholia
Delpierre G, Rider MH, Collard F, Stroobant V, Vanstapel F, Santos H, Van Schaftingen E.; ''Identification, cloning, and heterologous expression of a mammalian fructosamine-3-kinase.''; PubMedEurope PMCScholia
Butkowski RJ, Elion J, Downing MR, Mann KG.; ''Primary structure of human prethrombin 2 and alpha-thrombin.''; PubMedEurope PMCScholia
Zito E, Fraldi A, Pepe S, Annunziata I, Kobinger G, Di Natale P, Ballabio A, Cosma MP.; ''Sulphatase activities are regulated by the interaction of sulphatase-modifying factor 1 with SUMF2.''; PubMedEurope PMCScholia
Tie JK, Jin DY, Stafford DW.; ''Conserved loop cysteines of vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1) are involved in its active site regeneration.''; PubMedEurope PMCScholia
McMullen BA, Fujikawa K, Kisiel W, Sasagawa T, Howald WN, Kwa EY, Weinstein B.; ''Complete amino acid sequence of the light chain of human blood coagulation factor X: evidence for identification of residue 63 as beta-hydroxyaspartic acid.''; PubMedEurope PMCScholia
Selmer T, Hallmann A, Schmidt B, Sumper M, von Figura K.; ''The evolutionary conservation of a novel protein modification, the conversion of cysteine to serinesemialdehyde in arylsulfatase from Volvox carteri.''; PubMedEurope PMCScholia
Collard F, Wiame E, Bergans N, Fortpied J, Vertommen D, Vanstapel F, Delpierre G, Van Schaftingen E.; ''Fructosamine 3-kinase-related protein and deglycation in human erythrocytes.''; PubMedEurope PMCScholia
Schaffrath R, Abdel-Fattah W, Klassen R, Stark MJ.; ''The diphthamide modification pathway from Saccharomyces cerevisiae--revisited.''; PubMedEurope PMCScholia
Hirota Y, Tsugawa N, Nakagawa K, Suhara Y, Tanaka K, Uchino Y, Takeuchi A, Sawada N, Kamao M, Wada A, Okitsu T, Okano T.; ''Menadione (vitamin K3) is a catabolic product of oral phylloquinone (vitamin K1) in the intestine and a circulating precursor of tissue menaquinone-4 (vitamin K2) in rats.''; PubMedEurope PMCScholia
Vitamin K is a required co-factor in a single metabolic reaction, the gamma-carboxylation of glutamate residues of proteins catalyzed by GGCX (gamma-carboxyglutamyl carboxylase). Substrates of GGCX include blood clotting factors, osteocalcin (OCN), and growth arrest-specific protein 6 (GAS6) (Brenner et al. 1998). Vitamin K is derived from green leafy vegetables as phylloquinone and is synthesized by gut flora as menaquinone-7. These molecules are taken up by intestinal enterocytes with other lipids, packaged into chylomicrons, and delivered via the lymphatic and blood circulation to tissues of the body, notably hepatocytes and osteoblasts, via processes of lipoprotein trafficking (Shearer & Newman 2014; Shearer et al. 2012) described elsewhere in Reactome.
In these tissues, menadiol (reduced vitamin K3) reacts with geranylgeranyl pyrophosphate to form MK4 (vitamin K hydroquinone), the form of the vitamin required as cofactor for gamma-carboxylation of protein glutamate residues (Hirota et al. 2013). The gamma-carboxylation reactions, annotated elsewhere in Reactome as a part of protein metabolism, convert MK4 to its epoxide form, which is inactive as a cofactor. Two related enzymes, VKORC1 and VKORCL1, can each catalyze the reduction of MK4 epoxide to active MK4. VKORC1 activity is essential for normal operation of the blood clotting cascade and for osteocalcin function (Ferron et al. 2015). A physiological function for VKORCL1 has not yet been definitively established (Hammed et al. 2013; Tie et al. 2014).
Diphtheria is a serious, often fatal human disease associated with damage to many tissues. Bacteria in infected individuals, however, are typically confined to the lining of the throat or to a skin lesion; systemic effects are due to the secretion of an exotoxin encoded by a lysogenic bacteriophage. The toxin is encoded as a single polypeptide but is cleaved by host furin-like proteases to yield an aminoterminal fragment A and a carboxyterminal fragment B, linked by a disulfide bond. Toxin cleavage can occur when it first contacts the target cell surface, as annotated here, or as late as the point at which fragment A is released into the cytosol. Fragment B mediates toxin uptake into target cell endocytic vesicles, where acidification promotes a conformational change enabling fragment B to form a channel in the vesicle membrane through which fragment A is extruded into the target cell cytosol. Cleavage of the inter-fragment disulfide bond frees DT fragment A, which catalyzes ADP ribosylation of the translation elongation factor 2 (EEF2) in a target cell, thereby blocking protein synthesis. Neither fragment is toxic to human cells by itself (Collier 1975; Pappenheim 1977; Murphy 2011).
GGCX (gamma glutamyl carboxylase) in the endoplasmic reticulum gamma-carboxylates eleven glutamate residues on PROS1(25-676) (pro-protein S). MK4 (vitamin K hydroquinone) is oxidized to MK4 epoxide in the process (Berkner 2000; Furie et al. 1999; Stenina et al. 2001; Morris et al. 1995).
GGCX (gamma glutamyl carboxylase) in the endoplasmic reticulum gamma-carboxylates ten glutamate residues on F7(21-466) (pro-factor VII). MK4 (vitamin K hydroquinone) is oxidized to MK4 epoxide in the process (Berkner 2000; Furie et al. 1999; Stenina et al. 2001; Morris et al. 1995).
GGCX (gamma glutamyl carboxylase) in the endoplasmic reticulum gamma-carboxylates eight glutamate residues on 3D-PROC(33-197) (pro-protein C light chain). MK4 (vitamin K hydroquinone) is oxidized to MK4 epoxide in the process (Berkner 2000; Furie et al. 1999; Stenina et al. 2001; Morris et al. 1995).
GGCX (gamma glutamyl carboxylase) in the endoplasmic reticulum gamma-carboxylates twelve glutamate residues on 3D-F9(29-461) (pro-factor IX). MK4 (vitamin K hydroquinone) is oxidized to MK4 epoxide in the process (Berkner 2000; Furie et al. 1999; Stenina et al. 2001; Morris et al. 1995; Ware et al. 1989).
GGCX (gamma glutamyl carboxylase) in the endoplasmic reticulum gamma-carboxylates eleven glutamate residues on 3D-F10(32-179) (pro-factor X light chain). MK4 (vitamin K hydroquinone) is oxidized to MK4 epoxide in the process (Berkner 2000; Furie et al. 1999; Stenina et al. 2001; Morris et al. 1995).
GGCX (gamma glutamyl carboxylase) in the endoplasmic reticulum gamma-carboxylates ten glutamate residues on F2(25-622) (pro-prothrombin). MK4 (vitamin K hydroquinone) is oxidized to MK4 epoxide in the process (Berkner 2000; Furie et al. 1999; Stenina et al. 2001; Morris et al. 1995).
Sulfatase-modifying factor 2 (SUMF2, also called C-alpha-formylglycine-generating enzyme 2, pFGE) is the paralogue of SUMF1. While SUMF1 can modify a critical residue on arylsulfatases to confer activity to them, SUMF2 lacks this ability (Mariappan et al. 2005) and instead, SUMF2 can inhibit the action of SUMF1 by dimerising with it (Zito et al. 2005). SUMF2 can interact with sulfatases with and without SUMF1 (Zito et al. 2005).
The sulfatase-modifying factor 1 (SUMF1, also called C-alpha-formylglycine-generating enzyme, FGE) (Preusser-Kunze et al. 2005, Cosma et al. 2003, Landgrebe et al. 2003) oxidises the critical cysteine residue in arylsulfatases to an active site 3-oxoalanine residue thus confering sulfatase activity (Roeser et al. 2006). Defects in SUMF1 cause multiple sulfatase deficiency (MSD) (MIM:272200), an impairment of arylsulfatase activity due to defective post-translational modification of the cysteine residue (Cosma et al. 2003, Dierks et al, 2003). This post-translational modification is thought to be highly conserved in eukaryotes (Selmer et al. 1996, von Figura et al. 1998). SUMF1 is active as either a monomer or a homodimer. A monomer is described in this reaction.
GGCX (gamma glutamyl carboxylase) in the endoplasmic reticulum gamma-carboxylates eleven glutamate residues on GAS6(31-691) (pro-GAS6). MK4 (vitamin K hydroquinone) is oxidized to MK4 epoxide in the process. The details of the gamma-carboxylation of GAS6 have not been determined directly, but are inferred from those worked out for protein S (Manfioletti et al. 1993).
GGCX (gamma glutamyl carboxylase) in the endoplasmic reticulum gamma-carboxylates thirteen glutamate residues on PROZ(24-400) (pro-protein Z). MK4 (vitamin K hydroquinone) is oxidized to MK4 epoxide in the process (Berkner 2000; Furie et al. 1999; Stenina et al. 2001; Morris et al. 1995).
Cytosolic deoxyhypusine synthase (DHPS) tetramer catalyzes the reaction of the deoxyhypusine (Dhp) residue in EIF5A protein (Dhp-K50-EIF5A) with 1,3 diaminopropane, NADH and H+ to form EIF5A, spermidine (SPM), and NAD+ (Park et al. 2003; Park 2006). While this reaction is readily observed in vitro, it is probably minimized by the rapid, irreversible conversion of EIF5A Dhp residues to hypusine.
Cytosolic deoxyhypusine synthase (DHPS) tetramer catalyzes the reaction of of EIF5A protein, spermidine (SPM), and NAD+ to convert lysine 50 of EIF5A to deoxyhypusine (Dhp), generating 1,3 diaminopropane, NADH and H+ in the process (Clement et al. 2003; Joe et al. 1995; Park 2006; Wolff et al. 1997). Although the reaction is reversible, the reverse reaction is probably minimized under physiological conditions by the rapid, irreversible conversion of EIF5A Dhp residues to hypusine.
Cytosolic deoxyhypusine hydroxylase (DOHH) complexed with Fe2+ catalyzes the irreversible hydroxylation of peptidyl deoxyhypusine (Dhp-K50-EIF5A) to peptidyl hypusine (Hyp-K50-EIF5A) using molecular oxygen. The only known substrate for this enzyme is the modified lysine at residue 50 of the two isoforms of eIF5A (Clement et al. 2003; Kang et al. 2007; Kim et al. 2006).
Cytosolic diphthamide biosynthesis protein 6 (DPH6) ligates an ammonium ion to diphthine-EEF2 to generate diphthamide-EEF2 in a reaction coupled to the hydrolysis of ATP to yield AMP and PPi (Su et al. 2012; Uthman et al. 2013; Wei et al. 2013).
Cytosolic diphthamide biosynthesis protein 5 (DPH5) transfers four methyl groups from S-adenosylmethionine (AdoMet) to elongation factor 2 (EEF2) whose histidine residue at position 715 has been conjugated with a 3-amino 3-carboxypropyl group, forming methylated diphthine EEF2 and S-adenosylhomocysteine (AdoHcy). DPH5 activity has been identified in cells of diverse eukaryotic species including humans and has been characterized in detail in budding yeast (Liu et al. 2004; Matteakis et al. 1992; Moehring & Moehring 1988).
The diphthamide biosynthesis protein 2 (DPH2) subunit of the cytosolic DPH1:DPH2:DPH3 complex catalyzes the transfer of a 3-amino-3-carboxypropyl group from S-adenosylmethionine (AdoMet) to residue 715 of nascent elongation factor 2 (EEF2), forming aminocarboxypropyl EEF2 and S-methylthioadenosine (MTAD). The association of DPH1, 2, and 3 to form a complex is inferred from studies of the homologous yeast proteins (Abdel-Fattah et al. 2013; Bar et al. 2008) and more limited studies of interactions among mouse and human ones (Liu et al. 2004). The identification of DPH2 as the catalytically active subunit of the DPH1:DPH2:DPH3 complex is inferred from the properties of the homologous Pyrococcus horikoshii protein (Zhang et al. 2010). DPH4 (DNAJC24) is needed for the reaction to occur but its exact role is unknown (Liu et al. 2004; Su et al. 2013). DPH3 is an electron donor for DPH1-DPH2 in the first step of diphthamide biosynthesis (Dong et al. 2014).
By analogy to the activity of its experimentally characterized budding yeast homolog (Lin et al. 2014; Schaffrath et al. 2014), cytosolic DPH7 is inferred to catalyze the removal of a methyl group of Me-diphthine EEF2, yielding diphthine EEF2.
Protein-S-isoprenylcysteine O-methyltransferase (ICMT) mediates the post-translational methyl esterification of C-terminal CAAX motifs in prenylated proteins such as the oncoprotein RAS and related GTPases, neutralising the negative charge of prenylcysteine species and thereby determining their subcellular localisation and correct biological function (Wright et al. 2009, Yang et al. 2011). ICMT may serve as a therapeutic target in cancer development (Lau et al. 2014, Diver et al. 2014).
Proteins can undergo chemical modifications such as glycation, which occurs when glucose and other free aldoses spontaneously react with N-terminal and eta-amino groups of proteins to form Schiff bases, which slowly rearrange to ketosamines or, if the sugar was glucose, fructosamines. Fructosamines can further react slowly and become advanced glycation end products, which are thought to play a role in the pathophysiology of several disorders, especially diabetic complications. Ketosamine-3-kinase (FN3K) and ketosamine-3-kinase-related protein (FN3KRP) can phosphorylate protein-bound or free ketosamines on the third carbon of the sugar moiety and the resultant, unstable ketosamine 3-phosphates decompose under physiological conditions (a process called deglycation). Both enzymes can 3-phosphorylate psicosamines (PsiAm) and ribulosamines (RibAm) (Collard et al. 2003, 2004), but only FN3K can 3-phosphorylate fructosamines (FruAm) as well.
Proteins can undergo chemical modifications such as glycation, which occurs when glucose and other free aldoses spontaneously react with N-terminal and eta-amino groups of proteins to form Schiff bases, which slowly rearrange to ketosamines or, if the sugar is glucose, fructosamines. Fructosamines can further react slowly and become advanced glycation end products, which are thought to play a role in the pathophysiology of several disorders, especially diabetic complications. Ketosamine-3-kinase (FN3K) and ketosamine-3-kinase-related protein (FN3KRP) can phosphorylate protein-bound or free ketosamines on the third carbon of the sugar moiety and the resultant, unstable ketosamine 3-phosphates decompose under physiological conditions (a process called deglycation). Both enzymes can 3-phosphorylate psicosamines (PsiAm) and ribulosamines (RibAm), but only FN3K can 3-phosphorylate fructosamines (FruAm) as well (Delpierre et al. 2000, 2004).
GGCX (gamma glutamyl carboxylase) in the endoplasmic reticulum gamma-carboxylates three glutamate residues on BGLAP(24-100) (pro-osteocalcin). MK4 (vitamin K hydroquinone) is oxidized to MK4 epoxide in the process (Berkner 2000; Ferron et al. 2015; Furie et al. 1999; Hauschka et al. 1989; Morris et al. 1995; oser et al. 1980; Stenina et al. 2001).
Post-translational cellular processing of the factor VIII (FVIII or F8) precursor enables O-sulfation of tyrosine residues (Pittman DD et al. 1992; Michnick DA et al. 1994). Biochemical characterization demonstrated that recombinant human FVIII when metabolically labeled with [35S]-sulfate upon expression in Chinese hamster ovary (CHO) cells or monkey kidney tissue COS-1 cells contains six potential tyrosine sulfation sites, ie, four on the heavy chain (at amino acid residues 365, 737, 738, and 742) and two in the a3 subdomain of the light chain (residues 1683 and 1699) (Pittman DD et al. 1992; Michnick DA et al. 1994). The presence of six tyrosine sulfate residues in FVIII was further confirmed by a combination of liquid chromatography and electrospray ionization mass spectrometry (LC/ESI-MS) studies of the recombinant human FVIII protein derived from baby hamster kidney (BHK) cells (Severs JC et al. 1999) or CHO cells (Schmidbauer S et al. 2015). Site-directed mutagenesis of individual or multiple tyrosine residues showed that all the six sulfation sites are required to modulate FVIII activity (Pittman DD et al. 1992; Michnick DA et al. 1994). Further, treatment of CHO cells that express FVIII with sodium chlorate, an inhibitor of ATP sulphurylase involved in the synthesis of PAPS, did not affect FVIII secretion, but reduced the functional activity by 5-fold, indicating that sulfation was not required for FVIII secretion (Pittman DD et al. 1992). In addition, mutagenesis of Tyr1699 to Phe (Y1699F) demonstrated that sulfation at that residue was required for high affinity interaction of FVIII with von Willebrand factor (vWF) (Leyte A et al. 1991). In the absence of tyrosine sulfation at 1699 in FVIII, the affinity for vWF was reduced by 5-fold (Leyte A et al. 1991). The nuclear magnetic resonance (NMR) spectrum studies of the complex between FVIII and vWF showed significantly larger residue-specific chemical shift changes when Y1699 was sulfated further highlighting the importance of FVIII sulfation at Y1699 for the binding affinity to vWF (Dagil L et al. 2019). The significance of FVIII sulfation at Y1699 in vivo is made evident by the presence of a Y1699F mutation that causes a moderate hemophilia A, likely due to reduced interaction with vWF and decreased plasma half-life (Higuchi M et al. 1990; van den Biggelaar M et al. 2011). Sulfation at tyrosine residues 365 and 1683 increased FVIII activity by increasing the rate of thrombin cleavage at the adjacent thrombin cleavage sites 391 and 1708, respectively (Michnick DA et al. 1994). Mutation of tyrosine residues 737, 738, and 742 had no effect on the thromhin activation rate, even though the cleavage rate at Arg759 was slightly reduced (Michnick et al. 1994). Further. lower FXa-generation activity (86% of the wild-type activity) and lower clotting activity (51% of the wild-type activity) was observed for the FVIII triple point mutant at Tyr residues 737, 738, and 742 (Michnick et al. 1994). This result is in contrast to other study in which no functional differences were found between full-length FVIIl lacking sulfation at one or more of these three residues (Y737, Y738, and Y742) and the fully sulfated form of FVIII (Mikkelsen J et al. 1991).
Protein tyrosine O-sulfation is a common post-translational modification that is catalyzed by a tyrosyl protein sulfotransferase (TPST) (Moore KL 2003; Yang YS et al. 2015). In humans, two TPST isoforms, termed TPST1 and TPST2, have been identified (Ouyang Yb et al. 1998; Mishiro E et al. 2006). The enzyme was shown to catalyze the transfer of sulfate from the universal sulfate donor adenosine 3′-phosphate 5′-phosphosulfate (PAPS) to the hydroxyl group of a peptidyltyrosine residue to form a tyrosine O4-sulfate ester and 3′,5′-ADP (Lee RW & Huttner WB 1983). Structural studies showed that human TPSTs share the same catalytic mechanism (Teramoto T et al. 2013; Tanaka S et al. 2017). In mammalian cells, tyrosine O-sulfation of membrane and secretory proteins was found to occur in the trans-Golgi network, and biochemical studies indicated that the enzyme was membrane-bound (Lee RW & Huttner WB 1985; Baeuerle PA & Huttner WB 1987).
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In these tissues, menadiol (reduced vitamin K3) reacts with geranylgeranyl pyrophosphate to form MK4 (vitamin K hydroquinone), the form of the vitamin required as cofactor for gamma-carboxylation of protein glutamate residues (Hirota et al. 2013). The gamma-carboxylation reactions, annotated elsewhere in Reactome as a part of protein metabolism, convert MK4 to its epoxide form, which is inactive as a cofactor. Two related enzymes, VKORC1 and VKORCL1, can each catalyze the reduction of MK4 epoxide to active MK4. VKORC1 activity is essential for normal operation of the blood clotting cascade and for osteocalcin function (Ferron et al. 2015). A physiological function for VKORCL1 has not yet been definitively established (Hammed et al. 2013; Tie et al. 2014).
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Protein tyrosine O-sulfation is a common post-translational modification that is catalyzed by a tyrosyl protein sulfotransferase (TPST) (Moore KL 2003; Yang YS et al. 2015). In humans, two TPST isoforms, termed TPST1 and TPST2, have been identified (Ouyang Yb et al. 1998; Mishiro E et al. 2006). The enzyme was shown to catalyze the transfer of sulfate from the universal sulfate donor adenosine 3′-phosphate 5′-phosphosulfate (PAPS) to the hydroxyl group of a peptidyltyrosine residue to form a tyrosine O4-sulfate ester and 3′,5′-ADP (Lee RW & Huttner WB 1983). Structural studies showed that human TPSTs share the same catalytic mechanism (Teramoto T et al. 2013; Tanaka S et al. 2017). In mammalian cells, tyrosine O-sulfation of membrane and secretory proteins was found to occur in the trans-Golgi network, and biochemical studies indicated that the enzyme was membrane-bound (Lee RW & Huttner WB 1985; Baeuerle PA & Huttner WB 1987).