scholarly journals Heparin binding to platelet factor-4. An NMR and site-directed mutagenesis study: arginine residues are crucial for binding

1995 ◽  
Vol 312 (2) ◽  
pp. 357-365 ◽  
Author(s):  
K H Mayo ◽  
E Ilyina ◽  
V Roongta ◽  
M Dundas ◽  
J Joseph ◽  
...  
Keyword(s):  
Alpha Helix ◽  
Platelet Factor 4 ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Heparin Binding ◽  
Nmr Studies ◽  
Platelet Factor ◽  
Binding Data ◽  
Arginine Residues ◽  
Proton Resonances

Native platelet factor-4 (PF4) is an asymmetrically associated, homo-tetrameric protein (70 residues/subunit) known for binding polysulphated glycosaminoglycans like heparin. PF4 N-terminal chimeric mutant M2 (PF4-M2), on the other hand, forms symmetric tetramers [Mayo, Roongta, Ilyina, Milius, Barker, Quinlan, La Rosa and Daly (1995) Biochemistry 34, 11399-11409] making NMR studies with this 32 kDa protein tractable. PF4-M2, moreover, binds heparin with a similar affinity to that of native PF4. NMR data presented here indicate that heparin (9000 Da cut-off) binding to PF4-M2, while not perturbing the overall structure of the protein, does perturb specific side-chain proton resonances which map to spatially related residues within a ring of positively charged side chains on the surface of tetrameric PF4-M2. Contrary to PF4-heparin binding models which centre around C-terminal alpha-helix lysines, this study indicates that a loop containing Arg-20, Arg-22, His-23 and Thr-25, as well as Lys-46 and Arg-49, are even more affected by heparin binding. Site-directed mutagenesis and heparin binding data support these NMR findings by indicating that arginines more than C-terminal lysines, are crucial to the heparin binding process.

scholarly journals Identification and characterization of PF4varl, a human gene variant of platelet factor 4.

1989 ◽  
Vol 9 (4) ◽  
pp. 1445-1451 ◽  
Author(s):  
C J Green ◽  
R S Charles ◽  
B F Edwards ◽  
P H Johnson
Keyword(s):  
Amino Acid ◽  
Terminal Region ◽  
Leader Sequence ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Platelet Factor ◽  
Coding Sequences ◽  
Amino Terminal ◽  
Arginine Residues ◽  
Carboxy Terminal

A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissue-activating peptide III (two human platelet alpha-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variant of platelet factor 4, designated PF4var1. The gene for PF4var1 consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4var1 had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4var1 genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4var1. Rat PF4 and human PF4var1 genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.

Identification and characterization of PF4varl, a human gene variant of platelet factor 4

1989 ◽  
Vol 9 (4) ◽  
pp. 1445-1451
Author(s):  
C J Green ◽  
R S Charles ◽  
B F Edwards ◽  
P H Johnson
Keyword(s):  
Amino Acid ◽  
Terminal Region ◽  
Leader Sequence ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Platelet Factor ◽  
Coding Sequences ◽  
Amino Terminal ◽  
Arginine Residues ◽  
Carboxy Terminal

A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissue-activating peptide III (two human platelet alpha-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variant of platelet factor 4, designated PF4var1. The gene for PF4var1 consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4var1 had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4var1 genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4var1. Rat PF4 and human PF4var1 genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.

scholarly journals Purification of two heparin-binding proteins from porcine platelets and their homology with human secreted platelet proteins

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1072-1080 ◽  
Author(s):  
B Rucinski ◽  
A Poggi ◽  
P James ◽  
JC Holt ◽  
S Niewiarowski
Keyword(s):  
Amino Acid ◽  
Basic Protein ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Platelet Factor ◽  
Heterologous Antigens

Abstract Two heparin-neutralizing proteins secreted by thrombin-stimulated platelets were purified to homogeneity by means of heparin-agarose affinity chromatography. These proteins, termed porcine platelet basic protein (PBP) and porcine platelet factor 4 (PF4), were eluted from a heparin-agarose column at 0.6–0.9 M NaCl and at 1–1.4 M NaCl, respectively. The molecular weight of porcine platelet basic protein was 7,000–7,700 daltons, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid analysis. The isoelectric point of this protein was at pH 9.0. The amino acid composition of porcine platelet basic protein resembled that of human low affinity platelet factor 4 (LA-PF4), except that the porcine protein did not contain tyrosine. The molecular weight of porcine platelet factor 4 ranged from 10,000 (estimated from amino acid analysis) to 14,000 (estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis). The amino acid compositions of human platelet factor 4 and of porcine platelet factor 4 were similar. Monospecific antibodies against porcine platelet factor 4 and porcine platelet basic protein were raised in rabbits. Competitive radioimmunoassay demonstrated a low but significant immunologic cross-reactivity between human and porcine platelet factor 4, and between porcine platelet basic protein and a group of human secreted platelet proteins that bind to heparin with low affinity (beta-thromboglobulin [beta TG] and low affinity platelet factor 4). Experiments with direct immuno- precipitation of 125I-labeled antigens suggested that all four proteins investigated (human platelet factor 4, porcine platelet factor 4, human low affinity platelet factor 4 or human beta-thromboglobulin, and porcine platelet basic protein) share common antigenic determinants. However, there was a higher degree of immunologic cross-reactivity between heterologous antigens with similar heparin binding affinity (human platelet factor 4 and porcine platelet factor 4) than between heterologous antigens with different binding affinity (human platelet factor 4 and porcine platelet basic protein). In conclusion, our finding suggests a significant structural homology among the four proteins.

scholarly journals Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2

2002 ◽  
Vol 366 (3) ◽  
pp. 971-976 ◽  
Author(s):  
Lucimara CHIOATO ◽  
Arthur H.C. de OLIVEIRA ◽  
Roberto RULLER ◽  
Juliana M. SÁ ◽  
Richard J. WARD
Keyword(s):  
Phospholipase A2 ◽  
Lipid Membrane ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Phospholipid Bilayer ◽  
Directed Mutagenesis ◽  
Aromatic Residues ◽  
Loop Region ◽  
Terminal Loop ◽  
Arginine Residues

Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A2 from the venom of Bothrops jararacussu which demonstrates both myotoxic and Ca2+-independent membrane-damaging activities. The structural determinants of these activities are poorly defined, therefore site-directed mutagenesis has been used to substitute all cationic and aromatic residues between positions 115 and 129 in the C-terminal loop region of the protein. Substitution of lysine and arginine residues with alanine in the region 117—122 resulted in a significant reduction of myotoxic activity of the recombinant BthTx-I. With the exception of Lys122, these same substitutions did not significantly alter the Ca2+-independent membrane-damaging activity. In contrast, substitution of the positively-charged residues at positions 115, 116 and 122 resulted in reduced Ca2+-independent membrane-damaging activity but, with the exception of Lys122, had no effect on myotoxicity. These results indicate that the two activities are independent and are determined by discrete yet partially overlapping motifs in the C-terminal loop. Results from site-directed mutagenesis of the aromatic residues in the same part of the protein suggest that a region including residues 115—119 interacts superficially with the membrane interface and that the residues around position 125 partially insert into the lipid membrane. These results represent the first detailed mapping of a myotoxic site in a phospholipase A2, and support a model of a Ca2+-independent membrane-damaging mechanism in which the C-terminal region of BthTx-I interacts with and contributes to the perturbation of the phospholipid bilayer.

Methodologic Variations in the Determinations of Anti-Platelet Factor 4-Heparin Antibodies in Patients Suspected of Having Heparin Induced Thrombocytopenia: Diagnostic and Prognostic Implications.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3206-3206
Author(s):  
Jawed Fareed ◽  
Margaret Prechel ◽  
Michelle Kujawski ◽  
He Zhu ◽  
Jeanine Walenga ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Medical Center ◽  
Sandwich Elisa ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Diagnostic Efficacy ◽  
Diagnostic Reliability ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Two Samples

Abstract It is now widely believed that the pathogenesis of heparin induced thrombocytopenia (HIT) is largely mediated via the generation of anti-platelet factor 4-heparin antibodies (APFA) in patients who are treated with heparin and related drugs. In addition antibodies to other heparin binding proteins such as neutrophil activating peptide-2 and interleukins also contribute to this syndrome and which are not detectable by the methods based on heparin platelet factor 4 capture probes. Currently, several immunologic methods mostly utilizing sandwich ELISA techniques to measure APFA in sera of patients with HIT syndrome are available. These methods utilize different capture probes including heparin platelet factor 4 complex (ASSERACHROM® HPIA; Diagnostica Stago, France), platelet factor 4 complexed to polyvinyl sulfonate (PF4 Enhanced; GTI, USA) and heparin protamine suflate along with platelet lysates as a source of PF4 (ZYMUTEST HIA IgGAM; Hyphen Biomedical, France). These different capture probes for the APFA have different affinity for the APFAs. Moreover, these capture probes can also bind to certain other heparin binding proteins. To compare these three methods, samples (n = 100) were selected from banked sera that had been referred to Loyola University Medical Center for the quantitation of HIT antibodies and 14C Serotonin Release Assay (SRA). All of these specimens were initially positive in the GTI PF4 Enhanced ELISA for the presence of anti-heparin platelet factor 4 antibodies with a broad range of antibody titers absorbance range (0.4 – 2.5). For the head to head comparison all of these samples were assayed using the three different methods. In the GTI method the reassayed samples showed 93 out of 100 positive (93%), in the HPIA test 79 out of 100 (79%) and in the ZYMUTEST 56 out of 100 (56%) were positive. Interestingly, the correlation coefficients also showed marked variation (GTI vs ZYMUTEST r2 = 0.38; GTI vs Stago r2 = 0.49; ZYMUTEST vs Stago r2 = 0.67). The prevalence of positive samples was not consistent in the three tests used. This data clearly shows that each of the different ELISA methods exhibit different performance characteristics in binding to not only the APFA but other proteins, which may contribute to the higher false positive prevalence. Moreover, the positive/negative cutoff limits in these assays are arbitrarily set without due consideration of the antibody titer, which can also account for the observed differences. More interestingly, all of the samples positive in the SRA (n = 14) were positive in each of the three methods used with the exception of two samples, which were positive in the GTI, ASSERACHROM® HPIA and negative in the ZYMUTEST. These results clearly indicate the differences in the diagnostic efficacy of various commercially available tests and warrant clinical field trials to validate their reliability in the monitoring of APFA. Furthermore, the diagnostic reliability of these tests can be further improved by immunoglobulin subtyping and utilizing other capture probes with platelet factor 4 and related proteins, which complex with heparins.

scholarly journals Role of αArg145 and βArg263 in the active site of penicillin acylase of Escherichia coli

2002 ◽  
Vol 365 (1) ◽  
pp. 303-309 ◽  
Author(s):  
Wynand B.L. ALKEMA ◽  
Antoon K. PRINS ◽  
Erik de VRIES ◽  
Dick B. JANSSEN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Penicillin Acylase ◽  
Precursor Protein ◽  
Site Directed Mutagenesis ◽  
Penicillin G ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Arginine Residues

The active site of penicillin acylase of Escherichia coli contains two conserved arginine residues. The function of these arginines, αArg145 and βArg263, was studied by site-directed mutagenesis and kinetic analysis of the mutant enzymes. The mutants αArg145→Leu (αArg145Leu), αArg145Cys and αArg145Lys were normally processed and exported to the periplasm, whereas expression of the mutants βArg263Leu, βArg263Asn and βArg263Lys yielded large amounts of precursor protein in the periplasm, indicating that βArg263 is crucial for efficient processing of the enzyme. Either modification of both arginine residues by 2,3-butanedione or replacement by site-directed mutagenesis yielded enzymes with a decreased specificity (kcat/Km) for 2-nitro-5-[(phenylacetyl)amino]benzoic acid, indicating that both residues are important in catalysis. Compared with the wild type, the αArg145 mutants exhibited a 3–6-fold-increased preference for 6-aminopenicillanic acid as the deacylating nucleophile compared with water. Analysis of the steady-state parameters of these mutants for the hydrolysis of penicillin G and phenylacetamide indicated that destabilization of the Michaelis—Menten complex accounts for the improved activity with β-lactam substrates. Analysis of pH—activity profiles of wild-type enzyme and the βArg263Lys mutant showed that βArg263 has to be positively charged for catalysis, but is not involved in substrate binding. The results provide an insight into the catalytic mechanism of penicillin acylase, in which αArg145 is involved in binding of β-lactam substrates and βArg263 is important both for stabilizing the transition state in the reaction and for correct processing of the precursor protein.

scholarly journals Effect of Site-Directed Mutagenesis of Methylglyoxal-Modifiable Arginine Residues on the Structure and Chaperone Function of Human αA-Crystallin†

Biochemistry ◽  
2006 ◽  
Vol 45 (14) ◽  
pp. 4569-4577 ◽  
Author(s):  
Ashis Biswas ◽  
Antonia Miller ◽  
Tomoko Oya-Ito ◽  
Puttur Santhoshkumar ◽  
Manjunatha Bhat ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Chaperone Function ◽  
Arginine Residues
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