The Contributions of the P4-P2 Positions in PAR1 and 4 for Thrombin Recognition and Cleavage.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3574-3574
Author(s):  
Marvin T. Nieman ◽  
Alvin H. Schmaier
Keyword(s):  
Amino Acids ◽  
Cleavage Site ◽  
Receptor Activation ◽  
Breakdown Product ◽  
Wild Type ◽  
Binding Region ◽  
Alanine Scanning Mutagenesis ◽  
Important Amino Acid ◽  
Thrombin Cleavage

Abstract The angiotensin converting enzyme breakdown product of bradykinin, bradykinin 1–5 (RPPGF), inhibits thrombin induced human or mouse platelet aggregation by preventing proteolysis of PAR1 and PAR4 by binding adjacent to the thrombin cleavage site and thus preventing activation of these receptors (Am. J. Physiol.285:H183–H193, 2003; J. Pharmacol. Exp. Ther.311:492–501, 2004; FEBS Lett. 579:25–29, 2005). Alanine scanning mutagenesis determined that RPPGF binds to Pro46 at the P2 position of PAR4 to block thrombin cleavage. New studies determined the amino acids required for thrombin to bind and cleave PAR4. Wild type PAR4 exodomain is cleaved by thrombin with a Km of 17 μM, kcat of 3.5 s−1 and kcat/Km of 2x105 M−1 s−1. In contrast, PAR4 exodomain in which the P2 (PAR4-P46A) is changed to alanine, is not efficiently cleaved with 10 nM alpha thrombin. Alteration of PAR4’s P4 position (e.g., PAR4-P44A) does not influence the PAR4’s rate of cleavage. The ability of the PAR4 exodomain to inhibit thrombin proteolysis of H-D-Phe-Pip-pNA was also determined as an independent measure of the thrombin/PAR4 interaction. Wild type PAR4 exodomain and PAR4-P44A are competitive inhibitors of thrombin hydrolysis of the chromogenic substrate with a Ki of 23 ± 6 micromolar and 19.6 ± 4 μM respectively. In contrast, PAR4-P46A and PAR4-P44A/P46A have a Ki > 300 μM nd the nature of the inhibition changes from competitive to noncompetitive. Taken together, these data demonstrate that Pro46 of PAR4 is important for alpha thrombin to bind and orient PAR4 in its active site for efficient cleavage. Further studies examined the role of the combined amino acids in the P4 to P3 positions of PAR4 and PAR1 to contribute to the rate of thrombin cleavage. Chimeric molecules were prepared in which the P4 and P3 positions of PAR4 (ProAla) are replaced with those from PAR1 (LeuAsp) to generate PAR4-LD. The reciprocal chimera was also made (PAR1-PA). PAR4-LD is proteolyzed by 10 nM alpha thrombin more efficiently than PAR4-wt. Alternatively, the rate of PAR1-PA proteolysis by alpha thrombin is less efficient than PAR1-wt but better than PAR4-wt due to the presence of the exosite I binding region in the PAR1 exodomain. However, if the exosite I binding domain is removed from PAR1-PA the rate of cleavage is like that seen with PAR4-wt exodomain. These data indicate PAR1 is cleaved by alpha thrombin at a faster rate than PAR4 due to the amino acids in the P4 and P3 positions as well as the exosite I binding region. In sum, these data demonstrate that the P2 position of PAR4 is the most important amino acid in thrombin binding to the region adjacent to the thrombin cleavage site. However the combined P4 and P3 positions are also important determinants of the rate of receptor cleavage. These data indicate that the amino acids around the thrombin cleavage site of both PAR4 and 1 influence its rate of cleavage. Designing thrombin receptor activation antagonists directed to the thrombin cleavage site on PAR1 and 4 should be effective anti-platelet agents.

scholarly journals Immunologic analysis of the cloned platelet thrombin receptor activation mechanism: evidence supporting receptor cleavage, release of the N-terminal peptide, and insertion of the tethered ligand into a protected environment

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2125-2136 ◽  
Author(s):  
KJ Norton ◽  
RM Scarborough ◽  
JL Kutok ◽  
MA Escobedo ◽  
L Nannizzi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Cleavage Site ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Thrombin Receptor ◽  
Thrombin Cleavage ◽  
Thrombin Activation ◽  
Tethered Ligand

The recently cloned functional thrombin receptor is thought to be activated by thrombin cleavage of the bond between R41 and S42, followed by the insertion of the new N-terminal region (“tethered ligand”) into an unknown site in the receptor. Antibodies to peptides at or near the cleavage site have been reported to inhibit thrombin- induced platelet activation to varying extents, but the precise mechanism(s) of their inhibition is unknown. We have produced: (1) a polyclonal antibody in rabbits to a peptide containing amino acids 34 to 52 (anti-TR34–52); enzyme-linked immunosorbent assays (ELISA) indicate that anti-TR34–52 contains antibodies to regions on both sides of the thrombin cleavage site; (2) two murine monoclonal antibodies (MoAbs) to a peptide containing amino acids 29 to 68; one antibody reacts primarily with residues N-terminal to the thrombin cleavage site, and the other reacts primarily with residues C-terminal to the cleavage site; and (3) a polyclonal rabbit antibody to a peptide containing amino acids 83 to 94 (anti-TR83–94). Anti-TR34–52 binds to platelets as judged by flow cytometry, and pretreating platelets with a thrombin receptor peptide ligand does not lead to loss of antibody reactivity, suggesting that platelet activation does not initiate redistribution or internalization of surface thrombin receptors. In contrast, pretreating platelets with thrombin leads to complete loss of anti-TR34–52 binding. Similarly, the binding of both MoAbs to platelets is dramatically reduced by pretreatment with thrombin. However, the binding of anti-TR83–94 is not decreased by thrombin activation, confirming that the receptor is not internalized. Anti-TR34–52 profoundly inhibits low dose thrombin-induced platelet shape change and aggregation, but the inhibition can be overcome with higher thrombin doses. However, anti-TR34–52 does not inhibit platelet aggregation induced by tethered ligand peptides. The TR34–52 peptide is a thrombin substrate, with cleavage occurring at the R41-S42 bond as judged by high performance liquid chromatography (HPLC) and platelet aggregation analysis. Anti-TR34–52 prevented cleavage of the TR34–52 peptide, suggesting that the antibody prevents platelet activation, at least in part, by preventing cleavage of the thrombin receptor. These data, although indirect, provide additional support for a thrombin activation mechanism involving thrombin cleavage of the receptor; in addition, they provide new evidence indicating that receptor cleavage is followed by loss of the N-terminal peptide, and insertion of the tethered ligand into a protected domain.

scholarly journals Immunologic analysis of the cloned platelet thrombin receptor activation mechanism: evidence supporting receptor cleavage, release of the N-terminal peptide, and insertion of the tethered ligand into a protected environment

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2125-2136 ◽  
Author(s):  
KJ Norton ◽  
RM Scarborough ◽  
JL Kutok ◽  
MA Escobedo ◽  
L Nannizzi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Cleavage Site ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Thrombin Receptor ◽  
Thrombin Cleavage ◽  
Thrombin Activation ◽  
Tethered Ligand

Abstract The recently cloned functional thrombin receptor is thought to be activated by thrombin cleavage of the bond between R41 and S42, followed by the insertion of the new N-terminal region (“tethered ligand”) into an unknown site in the receptor. Antibodies to peptides at or near the cleavage site have been reported to inhibit thrombin- induced platelet activation to varying extents, but the precise mechanism(s) of their inhibition is unknown. We have produced: (1) a polyclonal antibody in rabbits to a peptide containing amino acids 34 to 52 (anti-TR34–52); enzyme-linked immunosorbent assays (ELISA) indicate that anti-TR34–52 contains antibodies to regions on both sides of the thrombin cleavage site; (2) two murine monoclonal antibodies (MoAbs) to a peptide containing amino acids 29 to 68; one antibody reacts primarily with residues N-terminal to the thrombin cleavage site, and the other reacts primarily with residues C-terminal to the cleavage site; and (3) a polyclonal rabbit antibody to a peptide containing amino acids 83 to 94 (anti-TR83–94). Anti-TR34–52 binds to platelets as judged by flow cytometry, and pretreating platelets with a thrombin receptor peptide ligand does not lead to loss of antibody reactivity, suggesting that platelet activation does not initiate redistribution or internalization of surface thrombin receptors. In contrast, pretreating platelets with thrombin leads to complete loss of anti-TR34–52 binding. Similarly, the binding of both MoAbs to platelets is dramatically reduced by pretreatment with thrombin. However, the binding of anti-TR83–94 is not decreased by thrombin activation, confirming that the receptor is not internalized. Anti-TR34–52 profoundly inhibits low dose thrombin-induced platelet shape change and aggregation, but the inhibition can be overcome with higher thrombin doses. However, anti-TR34–52 does not inhibit platelet aggregation induced by tethered ligand peptides. The TR34–52 peptide is a thrombin substrate, with cleavage occurring at the R41-S42 bond as judged by high performance liquid chromatography (HPLC) and platelet aggregation analysis. Anti-TR34–52 prevented cleavage of the TR34–52 peptide, suggesting that the antibody prevents platelet activation, at least in part, by preventing cleavage of the thrombin receptor. These data, although indirect, provide additional support for a thrombin activation mechanism involving thrombin cleavage of the receptor; in addition, they provide new evidence indicating that receptor cleavage is followed by loss of the N-terminal peptide, and insertion of the tethered ligand into a protected domain.

scholarly journals Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs

2021 ◽  
Author(s):  
Amit Ketkar ◽  
Lane Smith ◽  
Callie Johnson ◽  
Alyssa Richey ◽  
Makayla Berry ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mutation Frequency ◽  
Control Sequence ◽  
Wild Type ◽  
Binding Properties ◽  
High Affinity ◽  
G4 Dna ◽  
G Quadruplex ◽  
Forward Mutagenesis

Abstract We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

scholarly journals Deletion of 1–43 amino acids in cardiac myosin essential light chain blunts length dependency of Ca2+ sensitivity and cross-bridge detachment kinetics

2013 ◽  
Vol 304 (2) ◽  
pp. H253-H259 ◽  
Author(s):  
John Jeshurun Michael ◽  
Sampath K. Gollapudi ◽  
Steven J. Ford ◽  
Katarzyna Kazmierczak ◽  
Danuta Szczesna-Cordary ◽  
...  
Keyword(s):  
Amino Acids ◽  
Light Chain ◽  
Sarcomere Length ◽  
Cardiac Myosin ◽  
Wild Type ◽  
Essential Light Chain ◽  
Cross Bridge ◽  
Maximal Activation ◽  
Cardiac Muscle Fibers

The role of cardiac myosin essential light chain (ELC) in the sarcomere length (SL) dependency of myofilament contractility is unknown. Therefore, mechanical and dynamic contractile properties were measured at SL 1.9 and 2.2 μm in cardiac muscle fibers from two groups of transgenic (Tg) mice: 1) Tg-wild-type (WT) mice that expressed WT human ventricular ELC and 2) Tg-Δ43 mice that expressed a mutant ELC lacking 1–43 amino acids. In agreement with previous studies, Ca2+-activated maximal tension decreased significantly in Tg-Δ43 fibers. pCa50 (−log10 [Ca2+]free required for half maximal activation) values at SL of 1.9 μm were 5.64 ± 0.02 and 5.70 ± 0.02 in Tg-WT and Tg-Δ43 fibers, respectively. pCa50 values at SL of 2.2 μm were 5.70 ± 0.01 and 5.71 ± 0.01 in Tg-WT and Tg-Δ43 fibers, respectively. The SL-mediated increase in the pCa50 value was statistically significant only in Tg-WT fibers ( P < 0.01), indicating that the SL dependency of myofilament Ca2+ sensitivity was blunted in Tg-Δ43 fibers. The SL dependency of cross-bridge (XB) detachment kinetics was also blunted in Tg-Δ43 fibers because the decrease in XB detachment kinetics was significant ( P < 0.001) only at SL 1.9 μm. Thus the increased XB dwell time at the short SL augments Ca2+ sensitivity at short SL and thus blunts SL-mediated increase in myofilament Ca2+ sensitivity. Our data suggest that the NH2-terminal extension of cardiac ELC not only augments the amplitude of force generation, but it also may play a role in mediating the SL dependency of XB detachment kinetics and myofilament Ca2+ sensitivity.

DIRECTED MUTAGENESIS IN THE STUDY OF THE REQUIREMENTS FOR FACTOR VIII ACTIVITY IN VITRO AND IN VIVO

1987 ◽  
Author(s):  
Randal J Kaufman ◽  
Debra D Pittman ◽  
Louise C Wasley ◽  
W Barry Foster ◽  
Godfrey W Amphlett ◽  
...  
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Cleavage Site ◽  
Factor Xa ◽  
Cleavage Sites ◽  
Thrombin Cleavage ◽  
Terminal Fragment ◽  
Cofactor Activity

Factor VIII is a high molecular weight plasma glycoprotein that functions in the blood clotting cascade as the cofactor for factor DCa proteolytic activation of factor X. Factor VIII does not function proteolytically in this reaction hut itself can be proteolytically activated by other coagulation enzymes such as factor Xa and thrombin. In the plasma, factor VIII exists as a 200 kDa amino-terminal fragment in a metal ion stabilized complex with a 76 kDa carboxy-terminal fragment. The isolation of the cENA for human factor VIII provided the deduced primary amino acid sequence of factor VIIT and revealed three distinct structural domains: 1) a triplicated A domain of 330 amino acids which has homology to ceruloplasmin, a plasma copper binding protein, 2) a duplicated C domain of 150 amino acids, and 3) a unique B domain of 980 amino acids. These domains are arranged as shown below. We have previously reported the B domain is dispensible far cofactor activity in vitro (Toole et al. 1986 Proc. Natl. Acad 5939). The in vivo efficacy of factor VIII molecules harboring the B domain deletion was tested by purification of the wildtype and modified forms and infusion into factor VIII deficient, hemophilic, dogs. The wildtype and the deleted forms of recombinant derived factor VIII exhibited very similar survival curves (Tl/2 = 13 hrs) and the cuticle bleeding times suggested that both preparations appeared functionally equivalent. Sepharose 4B chromatography indicated that both factor VIII molecules were capable of binding canine plasma vWF.Further studies have addressed what cleavages are necessary for activation of factor VIII. The position of the thrombin, factor Xa, and activated protein C (AFC) cleavage sites within factor VIII are presented below, site-directed ENA medicated mutagenesis has been performed to modify the arginine at the amino side of each cleavagesite to an soleucine. In all cases this modification resulted in molecules that were resistant to cleavage by thrombin at the modified site. Modification of the thrombin cleavage sites at 336 and 740 and modification of the factor Xa cleavage site at 1721 resulted in no loss of cofactor activity. Modification of the thrombin cleavage site at either 372 or 1689 destroyed oofactor activity. Modification of the thrombin cleavage site at 336 resulted in a factor VIII having an increased activity, possibly due to resistance to inactivation. These results suggest the requirement of cleavage at residues 372 and 1689 for cofactor activity.

Negative Effects of GM-CSF Signaling In t(8;21)-Induced Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3163-3163
Author(s):  
Shinobu Matsuura ◽  
Ming Yan ◽  
Eun-Young Ahn ◽  
Miao-Chia Lo ◽  
David Dangoor ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Sex Chromosome ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Receptor Activation ◽  
Pseudoautosomal Region ◽  
Wild Type ◽  
Gm Csf

Abstract Abstract 3163 The t(8;21)(q22;q22) translocation is one of the most common chromosomal translocations in de novo acute myeloid leukemia (AML). The 8;21 translocation is often associated with additional cytogenetic abnormalities. The loss of the sex chromosome (LOS) is by far the most frequent abnormality found in association with the t(8;21) leukemia, accounting for 32–59% of patients, in contrast to other types of AML in which the LOS occurs in less than 5% of patients. To evaluate the role of sex chromosome deletion in t(8;21)-related leukemogenesis, hematopoietic cells from a mouse line with only one sex chromosome were used in retrovirus-mediated t(8;21) (AML1-ETO) expression and transplantation assays. The absence of leukemia in those animals suggested that a gene present in the pseudoautosomal region of sex chromosomes in humans but not in mice may be the target gene in LOS. The granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) gene is one such gene and is also known to be involved in myeloid cell survival, proliferation and differentiation. The GM-CSFRα gene is specifically down-regulated in AML patients with t(8;21), but not in other common translocations (Valk PJM et al, NEJM, 2004). The GM-CSFR complex is composed of α and βc subunits that assemble into a complex for receptor activation and signaling. To investigate the role of GM-CSFR signaling in t(8;21)-mediated leukemogenesis, GM-CSFR common β subunit knockout (GM-CSFRβc-/-) mice were used in our studies as a model for deficient GM-CSFR signaling. Transduction of AML1-ETO in hematopoietic cells from GM-CSFRβc-/- resulted in myeloid leukemia of a median survival time of 225 days, high percentage of blasts in peripheral blood and bone marrow, anemia, thrombocytopenia, hepatomegaly and splenomegaly. Comparison of wild-type and GM-CSFRβc-/- cells in the same transplantation resulted in development of AML1-ETO-induced leukemia at higher penetrance in GM-CSFRβc-/- cells (28.5% vs 100%). Moreover, the latency of leukemia was shorter in GM-CSFRβc-/- cells than in wild-type cells after transduction of AML1-ETO9a. Analysis of the hematopoietic compartment of healthy GM-CSFRβc-/- mice detected no significant abnormalities in the immature hematopoietic compartment (LSK, CMP, GMP, MEP), suggesting that AML1-ETO expression is required for leukemia to occur. In vitro, expression of AML1-ETO alone is sufficient for the immortalization of normal hematopoietic cells, as demonstrated by serial replating capacity of cells in methylcellulose colony assay. Addition of mGM-CSF to the basic cytokine cocktail (mIL-3, hIL-6, mSCF, hEPO) did not significantly affect number, type, size, and cell composition of colony cells. In contrast, the addition of mGM-CSF eliminated the replating capacity of AML1-ETO expressing cells, although they survived longer than control vector-infected cells. The results suggest that activation of GM-CSF signaling can specifically abrogate the self-renewal ability of potential leukemic stem cells in the early immortalization phase. These results support a possible tumor suppressor role of GM-CSF in leukemogeneis by AML1-ETO and may provide clues to understand how AML1-ETO corrupts normal GM-CSF signals to its own advantage for leukemogenic transformation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hydrophilic Domain II of Escherichia coli Dr Fimbriae Facilitates Cell Invasion

2005 ◽  
Vol 73 (9) ◽  
pp. 6119-6126 ◽  
Author(s):  
Margaret Das ◽  
Audrey Hart-Van Tassell ◽  
Petri T. Urvil ◽  
Susan Lea ◽  
David Pettigrew ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Cell Invasion ◽  
Cell Entry ◽  
Wild Type ◽  
Alanine Scanning Mutagenesis ◽  
Decay Accelerating Factor ◽  
Fimbrial Subunit ◽  
Hydrophilic Domain ◽  
Intracellular Compartmentalization

ABSTRACT Uropathogenic and diarrheal Escherichia coli strains expressing adhesins of the Dr family bind to decay-accelerating factor, invade epithelial cells, preferentially infect children and pregnant women, and may be associated with chronic or recurrent infections. Thus far, no fimbrial domain(s) that facilitates cell invasion has been identified. We used alanine scanning mutagenesis to replace selected amino acids in hydrophilic domain II of the structural fimbrial subunit DraE and evaluated recombinant mutant DraE for attachment, invasion, and intracellular compartmentalization. The mutation of amino acids V28, T31, G33, Q34, T36, and P40 of DraE reduced or abolished HeLa cell invasion but did not affect attachment. Electron micrographs showed a stepwise entry and fusion of vacuoles containing Escherichia coli mutants T36A and Q34A or corresponding beads with lysosomes, whereas vacuoles with wild-type Dr adhesin showed no fusion. Mutants T31A and Q34A, which were deficient in invasion, appeared to display a reduced capacity for clustering decay-accelerating factor. Our findings suggest that hydrophilic domain II may be involved in cell entry. These data are consistent with the interpretation that in HeLa cells the binding and invasion phenotypes of Dr fimbriae may be separated.

Arginine 595 is duplicated in patients with acute leukemias carrying internal tandem duplications of FLT3 and modulates its transforming potential

Blood ◽  
2007 ◽  
Vol 110 (2) ◽  
pp. 686-694 ◽  
Author(s):  
Sridhar Vempati ◽  
Carola Reindl ◽  
Seshu Kumar Kaza ◽  
Ruth Kern ◽  
Theodora Malamoussi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Mechanism ◽  
Positive Charge ◽  
Critical Role ◽  
Heterogeneous Group ◽  
Wild Type ◽  
Acute Leukemias ◽  
Juxtamembrane Region ◽  
Tandem Duplications

Abstract FLT3–internal tandem duplications (FLT3-ITDs) comprise a heterogeneous group of mutations in patients with acute leukemias that are prognostically important. To characterize the mechanism of transformation by FLT3-ITDs, we sequenced the juxtamembrane region (JM) of FLT3 from 284 patients with acute leukemias. The length of FLT3-ITDs varied from 2 to 42 amino acids (AAs) with a median of 17 AAs. The analysis of duplicated AAs showed that in the majority of patients, the duplications localize between AAs 591 to 599 (YVDFREYEY). Arginine 595 (R595) within this region is duplicated in 77% of patients. Single duplication of R595 in FLT3 conferred factor-independent growth to Ba/F3 cells and activated STAT5. Moreover, deletion or substitution of the duplicated R595 in 2 FLT3-ITD constructs as well as the deletion of wild-type R595 in FLT3-ITD substantially reduced the transforming potential and STAT5 activation, pointing to a critical role of the positive charge of R595 in stabilizing the active confirmation of FLT3-ITDs. Deletion of R595 in FLT3-WT nearly abrogated the ligand-dependent activation of FLT3-WT. Our data provide important insights into the molecular mechanism of transformation by FLT3-ITDs and show that duplication of R595 is important for the leukemic potential of FLT3-ITDs.

Human acetyl-CoA:glucosamine-6-phosphate N-acetyltransferase 1 has a relaxed donor specificity and transfers acyl groups up to four carbons in length

2016 ◽  
Vol 94 (2) ◽  
pp. 197-204 ◽  
Author(s):  
Inka Brockhausen ◽  
Dileep G. Nair ◽  
Min Chen ◽  
Xiaojing Yang ◽  
John S. Allingham ◽  
...  
Keyword(s):  
Amino Acids ◽  
Articular Cartilage ◽  
Enzymatic Synthesis ◽  
Biomedical Applications ◽  
Binding Pocket ◽  
Wild Type ◽  
Acetyl Coenzyme A ◽  
Site Specific ◽  
Acetyl Group

Glucosamine-6-phosphate N-acetyltransferase1 (GNA1) catalyses the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to glucosamine-6-phosphate (GlcN6P) to form N-acetylglucosamine-6-phosphate (GlcNAc6P), which is an essential intermediate in UDP-GlcNAc biosynthesis. An analog of GlcNAc, N-butyrylglucosamine (GlcNBu) has shown healing properties for bone and articular cartilage in animal models of arthritis. The goal of this work was to examine whether GNA1 has the ability to transfer a butyryl group from butyryl-CoA to GlcN6P to form GlcNBu6P, which can then be converted to GlcNBu. We developed fluorescent and radioactive assays and examined the donor specificity of human GNA1. Acetyl, propionyl, n-butyryl, and isobutyryl groups were all transferred to GlcN6P, but isovaleryl-CoA and decanoyl-CoA did not serve as donor substrates. Site-specific mutants were produced to examine the role of amino acids potentially affecting the size and properties of the AcCoA binding pocket. All of the wild type and mutant enzymes showed activities of both acetyl and butyryl transfer and can therefore be used for the enzymatic synthesis of GlcNBu for biomedical applications.

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