Cloning and characterization of platelet factor 4 cDNA derived from a human erythroleukemic cell line

Abstract We report the isolation of a platelet factor 4 (PF4) cDNA clone from a lambda gt11 expression cDNA library which was derived from a human erythroleukemic (HEL) cell line. The sequence of the DNA insert includes the 3′-untranslated region, the entire amino acid coding region for the mature PF4 protein, and a 5′ region containing coding information for an additional 18 amino acids. In addition, supplemental genomic DNA sequencing shows that the full-length leader sequence is 30 amino acids long plus an initial methionine and codes for a hydrophobic signal-like sequence which is probably involved in transmembrane transport. A single species mRNA of approximately 800 nucleotides was detected on blots of HEL cell poly(A) + RNA using a labeled PF4 cDNA probe. The human PF4 leader sequence shares some DNA, but no amino acid, homology with the 15 amino acids at the N-terminus of mature bovine PF4, suggesting rapid divergence in this region of PF4 between these two species. Sequence comparison of the coding regions of mature PF4 and gamma IP-10, a protein induced in a variety of cells following treatment with gamma-interferon, shows a corrected divergence of 76%. The divergence of a common ancestor protein into PF4 and gamma IP-10 may have accompanied the development of sophisticated immune and coagulation systems in vertebrates. The availability of cDNA and genomic DNA information for these genes in other species will be useful in studying the evolution of the coagulation and immune systems.

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Cloning and characterization of platelet factor 4 cDNA derived from a human erythroleukemic cell line

Blood ◽

10.1182/blood.v69.1.219.bloodjournal691219 ◽

1987 ◽

Vol 69 (1) ◽

pp. 219-223 ◽

Cited By ~ 5

Author(s):

M Poncz ◽

S Surrey ◽

P LaRocco ◽

MJ Weiss ◽

EF Rappaport ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Cell Line ◽

Genomic Dna ◽

Cdna Probe ◽

Leader Sequence ◽

Platelet Factor 4 ◽

Coding Region ◽

Platelet Factor ◽

Amino Acid Homology

We report the isolation of a platelet factor 4 (PF4) cDNA clone from a lambda gt11 expression cDNA library which was derived from a human erythroleukemic (HEL) cell line. The sequence of the DNA insert includes the 3′-untranslated region, the entire amino acid coding region for the mature PF4 protein, and a 5′ region containing coding information for an additional 18 amino acids. In addition, supplemental genomic DNA sequencing shows that the full-length leader sequence is 30 amino acids long plus an initial methionine and codes for a hydrophobic signal-like sequence which is probably involved in transmembrane transport. A single species mRNA of approximately 800 nucleotides was detected on blots of HEL cell poly(A) + RNA using a labeled PF4 cDNA probe. The human PF4 leader sequence shares some DNA, but no amino acid, homology with the 15 amino acids at the N-terminus of mature bovine PF4, suggesting rapid divergence in this region of PF4 between these two species. Sequence comparison of the coding regions of mature PF4 and gamma IP-10, a protein induced in a variety of cells following treatment with gamma-interferon, shows a corrected divergence of 76%. The divergence of a common ancestor protein into PF4 and gamma IP-10 may have accompanied the development of sophisticated immune and coagulation systems in vertebrates. The availability of cDNA and genomic DNA information for these genes in other species will be useful in studying the evolution of the coagulation and immune systems.

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Complete Covalent Structure of Human Platelet Factor 4

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657071 ◽

1979 ◽

Vol 42 (05) ◽

pp. 1652-1660 ◽

Cited By ~ 4

Author(s):

Francis J Morgan ◽

Geoffrey S Begg ◽

Colin N Chesterman

Keyword(s):

Amino Acids ◽

Molecular Weight ◽

Amino Acid ◽

Amino Acid Sequence ◽

Human Platelet ◽

Platelet Factor 4 ◽

Platelet Factor ◽

Disulphide Bonds ◽

Covalent Structure

SummaryThe amino acid sequence of the subunit of human platelet factor 4 has been determined. Human platelet factor 4 consists of identical subunits containing 70 amino acids, each with a molecular weight of 7,756. The molecule contains no methionine, phenylalanine or tryptophan. The proposed amino acid sequence of PF4 is: Glu-Ala-Glu-Glu-Asp-Gly-Asp-Leu-Gln-Cys-Leu-Cys-Val-Lys-Thr-Thr-Ser- Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Pro-Thr-Ala-Gin- Leu-Ile-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Leu-Gln-Ala-Pro-Leu-Tyr-Lys-Lys- Ile-Ile-Lys-Lys-Leu-Leu-Glu-Ser. From consideration of the homology with p-thromboglobulin, disulphide bonds between residues 10 and 36 and between residues 12 and 52 can be inferred.

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

Molecular and Cellular Biology ◽

10.1128/mcb.9.4.1445 ◽

1989 ◽

Vol 9 (4) ◽

pp. 1445-1451 ◽

Cited By ~ 38

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.

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Nomenclature of Secreted Platelet Proteins – Report of the Working Party on Secreted Platelet Proteins of the Subcommittee on Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661294 ◽

1985 ◽

Vol 53 (02) ◽

pp. 282-284 ◽

Cited By ~ 10

Author(s):

Karen L Kaplan ◽

Stefan Niewiarowski

Keyword(s):

Amino Acids ◽

Molecular Weight ◽

Amino Acid ◽

Cellulose Acetate ◽

Working Party ◽

Platelet Factor 4 ◽

Cellulose Acetate Electrophoresis ◽

Platelet Factor ◽

Amino Terminal ◽

Platelet Proteins

SummaryStandard nomenclature for a number of secreted platelet proteins was agreed upon by The Working Party on Secreted Platelet Proteins of the Subcommittee on Platelets. Platelet factor 4 will continue to be used for the molecule with high heparin affinity, subunit molecular weight of 7780, and the described amino acid sequence. β-Thromboglobulin will be used to designate β-Thromboglobulin (81 amino acids/subunit, β-mobility on cellulose-acetate electrophoresis, pI 7), low-affinity platelet factor 4 (85 amino acids/subunit, y-mobility on cellulose-acetate electrophoresis, pI 8), and platelet basic protein (94 amino acids/ subunit, pI 10) when these are measured immunologically in plasma, but that thromboglobulin with a superscript designation of the pI should be used when assays are conducted on samples after isoelectric focusing, and a subscript amino-terminal amino acid can be added when a purified protein is described. Thrombospondin will continue to be the designation for the high molecular weight trimer that has previously been called thrombospondin or glycoprotein G. Platelet derived growth factor will be used for the group of closely related proteins of molecular weight about 30,000 and isoelectric point about 10.

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

Molecular and Cellular Biology ◽

10.1128/mcb.9.4.1445-1451.1989 ◽

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

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Murine erythropoietin gene: cloning, expression, and human gene homology.

Molecular and Cellular Biology ◽

10.1128/mcb.6.3.849 ◽

1986 ◽

Vol 6 (3) ◽

pp. 849-858 ◽

Cited By ~ 131

Author(s):

C B Shoemaker ◽

L D Mitsock

Keyword(s):

Amino Acid ◽

Genomic Library ◽

Cdna Probe ◽

Amino Acid Sequences ◽

Nucleotide Sequence Analysis ◽

Coding Region ◽

Transcription Control ◽

Erythroid Progenitor ◽

Human Genes ◽

Cell Expression

The gene for murine erythropoietin (EPO) was isolated from a mouse genomic library with a human EPO cDNA probe. Nucleotide sequence analysis permitted the identification of the murine EPO coding sequence and the prediction of the encoded amino acid sequence based on sequence conservation between the mouse and human EPO genes. Both the coding DNA and the amino acid sequences were 80% conserved between the two species. Transformation of COS-1 cells with a mammalian cell expression vector containing the murine EPO coding region resulted in secretion of murine EPO with biological activity on both murine and human erythroid progenitor cells. The transcription start site for the murine EPO gene in kidneys was determined. This permitted tentative identification of the transcription control region. The region included 140 base pairs upstream of the cap site which was over 90% conserved between the murine and human genes. Surprisingly, the first intron and much of the 5'- and 3'-untranslated sequences were also substantially conserved between the genes of the two species.

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Structure of the rat platelet factor 4 gene: a marker for megakaryocyte differentiation.

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.898 ◽

1987 ◽

Vol 7 (2) ◽

pp. 898-904 ◽

Cited By ~ 61

Author(s):

T Doi ◽

S M Greenberg ◽

R D Rosenberg

Keyword(s):

Amino Acid ◽

Sequence Analysis ◽

Genomic Library ◽

Transcriptional Start Site ◽

Platelet Factor 4 ◽

Start Codon ◽

Cdna Expression Library ◽

Amino Acid Residues ◽

Platelet Factor ◽

Nuclease Mapping

A rat platelet factor 4 (PF4) cDNA has been isolated by immunoscreening a g lambda 11 rat megakaryocyte cDNA expression library. Sequence analysis of the rat PF4 cDNA revealed that this megakaryocyte protein is composed of a leader sequence of 29 amino acid residues and a mature protein sequence of 76 amino acid residues. The structure of rat PF4 derived from its cDNA shows a marked homology with the amino acid sequence of human PF4 obtained by classical protein chemistry techniques. This observation is particularly striking with regard to the carboxy-terminal region of rat and human PF4, where 28 of the last 31 C-terminal residues are identical. The rat PF4 gene was obtained from a rat genomic library by using rat PF4 cDNA as a hybridization probe. Sequence analysis showed that the gene is constructed of three exons and two short introns. The transcriptional start site is located 73 base pairs upstream of the translational start codon as judged by S1 nuclease mapping and primer extension. The 5' noncoding region of the gene also exhibited a sequence homologous to the TATA box at -31, as well as a series of direct and inverted repeat sequences and a cluster of 26 T residues at -155 to -218. This latter domain may be involved in regulating PF4 gene expression during megakaryocytopoiesis.

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Multiple pathways for cationic amino acid transport in rat thyroid epithelial cell line PC Cl3

AJP Cell Physiology ◽

10.1152/ajpcell.00053.2004 ◽

2005 ◽

Vol 288 (2) ◽

pp. C290-C303 ◽

Cited By ~ 10

Author(s):

Tiziano Verri ◽

Cinzia Dimitri ◽

Sonia Treglia ◽

Fabio Storelli ◽

Stefania De Micheli ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Cell Line ◽

Amino Acid Transport ◽

Transport Systems ◽

Residual Fraction ◽

Acid Transport ◽

Thyroid Cells ◽

Cationic Amino Acid ◽

Rat Thyroid

Information regarding cationic amino acid transport systems in thyroid is limited to Northern blot detection of y+LAT1 mRNA in the mouse. This study investigated cationic amino acid transport in PC cell line clone 3 (PC Cl3 cells), a thyroid follicular cell line derived from a normal Fisher rat retaining many features of normal differentiated follicular thyroid cells. We provide evidence that in PC Cl3 cells plasmalemmal transport of cationic amino acids is Na+ independent and occurs, besides diffusion, with the contribution of high-affinity, carrier-mediated processes. Carrier-mediated transport is via y+, y+L, and b0,+ systems, as assessed by l-arginine uptake and kinetics, inhibition of l-arginine transport by N-ethylmaleimide and neutral amino acids, and l-cystine transport studies. y+L and y+ systems account for the highest transport rate (with y+L > y+) and b0,+ for a residual fraction of the transport. Uptake data correlate to expression of the genes encoding for CAT-1, CAT-2B, 4F2hc, y+LAT1, y+LAT2, rBAT, and b0,+AT, an expression profile that is also shown by the rat thyroid gland. In PC Cl3 cells cationic amino acid uptake is under TSH and/or cAMP control (with transport increasing with increasing TSH concentration), and upregulation of CAT-1, CAT-2B, 4F2hc/y+LAT1, and rBAT/b0,+AT occurs at the mRNA level under TSH stimulation. Our results provide the first description of an expression pattern of cationic amino acid transport systems in thyroid cells. Furthermore, we provide evidence that extracellular l-arginine is a crucial requirement for normal PC Cl3 cell growth and that long-term l-arginine deprivation negatively influences CAT-2B expression, as it correlates to reduction of CAT-2B mRNA levels.

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Two site-directed mutations abrogate enzyme activity but have different effects on the conformation and cellular content of the N-acetylgalactosamine 4-sulphatase protein

Biochemical Journal ◽

10.1042/bj3070457 ◽

1995 ◽

Vol 307 (2) ◽

pp. 457-463 ◽

Cited By ~ 24

Author(s):

D A Brooks ◽

D A Robertson ◽

C Bindloss ◽

T Litjens ◽

D S Anson ◽

...

Keyword(s):

Amino Acids ◽

Enzyme Activity ◽

Amino Acid ◽

Cell Line ◽

Protein Level ◽

Structural Modification ◽

Wild Type ◽

Cellular Content ◽

Control Protein ◽

Wild Type Control

The sulphatase family of enzymes have regions of sequence similarity, but relatively little is known about either the structure-function relationships of sulphatases, or the role of highly conserved amino acids. The sequence of amino acids CTPSR at position 91-95 of 4-sulphatase has been shown to be highly conserved in all of the sequenced sulphatase enzymes. The cysteine at amino acid 91 of 4-sulphatase was selected for mutation analysis due to its potential role in either the active site, substrate-binding site or part of a key structural domain of 4-sulphatase and due to the absence of naturally occurring mutations in this residue in mucopolysaccharidosis type VI (MPS VI) patients. Two mutations, C91S and C91T, altering amino acid 91 of 4-sulphatase were generated and expressed in Chinese hamster ovary cells. Biochemical analysis of protein from a C91S cell line demonstrated no detectable 4-sulphatase enzyme activity but a relatively normal level of 4-sulphatase polypeptide (180% of the wild-type control protein level). Epitope detection, using a panel of ten monoclonal antibodies, demonstrated that the C91S polypeptide had a similar immunoreactivity to wild-type 4-sulphatase, suggesting that the C91S substitution does not induce a major structural change in the protein. Reduced catalytic activity associated with normal levels of 4-sulphatase protein have not been observed in any of the MPS VI patients tested and all show evidence of structural modification of 4-sulphatase protein with the same panel of antibodies [Brooks, McCourt, Gibson, Ashton, Shutter and Hopwood (1991) Am. J. Hum. Genet. 48, 710-719]. The loss of enzyme activity without a detectable protein conformation change suggests that Cys-91 may be a critical residue in the catalytic process. In contrast, analysis of protein from a C91T cell line revealed low levels of catalytically inactive 4-sulphatase polypeptide (0.37% of the wild-type control protein level) which had missing or masked epitopes, suggesting an altered protein structure or conformation. Subcellular fractionation studies of the C91T cell line demonstrated a high proportion of 4-sulphatase polypeptide content in organelles characteristic of microsomes. The aberrant intracellular localization and the reduced cellular content of 4-sulphatase polypeptide was consistent with the observed structural modification leading to retention and degradation of the protein within an early vacuolar compartment.

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

Blood ◽

10.1182/blood.v61.6.1072.1072 ◽

1983 ◽

Vol 61 (6) ◽

pp. 1072-1080 ◽

Cited By ~ 15

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.

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