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Colvin M Redman

Publications and source records attributed to Colvin M Redman.

8 recordsLinked to original sources
PubMed2006

Identification of two new members, XPLAC and XTES, of the XK family.

XK, a putative membrane transporter, is a component of the XK/Kell complex of the Kell blood group system. XK's substrate is unknown but absence of the protein, as occurs in the McLeod phenotype, is associated with red cell acanthocytosis and late onset central nervous system and neuromuscular abnormalities known as the McLeod syndrome. We have cloned two cDNAs, XPLAC (GenBank accession no. AY589511) and XTES (GenBank accession no. AY989815), which are closely related to XK and define them together as the XK family. XPLAC has a 2.9 kb cDNA that encodes 462 amino acids and XTES has a 1.6 kb cDNA coding 459 amino acids. The predicted molecular weights are 53.6 kDa for XPLAC and 53.4 kDa for XTES, which are similar to that of XK, which is 50.9 kDa. Unlike XK which is ubiquitously expressed XPLAC is expressed mostly in placenta and adrenal gland while XTES is exclusively expressed in primate testis. XPLAC has 37% and XTES has 31% amino acid identity with XK protein and they are predicted to have a similar topology to XK. XPLAC, like XK, has 3 exons and is located on X chromosome at q22.1, while XTES has 4 exons and is located at 22q11.1. Phylogenetic analysis shows that there are at least 5 additional vertebrate genes that are evolutionarily distantly related to the XK family. A domain with consensus sequences (ced-8 domain) for the extended family is described.

Acanthocytes↗
PubMed2006

Endothelin-3-converting enzyme activity of the KEL1 and KEL6 phenotypes of the Kell blood group system.

The Kell blood group protein is a metalloendopeptidase that preferentially cleaves a Trp(21)-Ile(22) bond of big endothelin-3 producing bioactive endothelin-3. Kell is a polymorphic protein, and 25 different phenotypes, because of point mutations resulting in single amino acid substitutions, have been described. It was recently reported that a recombinant form of KEL1 (K, K1) phenotype, expressed in K562 and HEK293 cells, had no endothelin-3-converting activity, in contrast to the common KEL2 (k, K2) phenotype. We demonstrate that KEL1 red blood cells and also a soluble recombinant form of KEL1 protein (s-Kell KEL1) have similar enzymatic activity as the common Kell phenotype. In addition we show that KEL6 red blood cells, which are more prevalent in persons of African heritage than in Caucasians also have endothelin-3-converting enzyme activity and that the recombinant soluble form of KEL6 protein (s-Kell KEL6) has similar K(m) values as the wild-type.

Animals↗
PubMed2005

Onset of expression of the components of the Kell blood group complex.

BACKGROUND: Kell and XK, two distinct red blood cell membrane proteins, are linked by a disulfide bond and form the Kell blood group complex. Kell surface antigens are expressed early during erythropoiesis but the onset of expression of XK which carries the Kx antigen is unknown. STUDY DESIGN AND METHODS: To determine whether Kell and XK are synchronously expressed, sorted human hematopoietic progenitor cells and mouse progenitor cells of defined lineage were studied. To determine the onset of expression, human marrow and cord blood cells were sorted into three subpopulations, representing stem, multipotent, and erythroid progenitor cells, and the expression of Kell and XK was determined by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorting (FACS) analysis. Mouse Kell and XK transcripts were determined by cDNA blotting of progenitor cells of defined lineage. RESULTS: By RT-PCR, human peripheral blood progenitor cells had weak expression of Kell and XK transcripts but FACS analysis did not detect surface antigens. Kell and XK transcripts are expressed in multipotent progenitor cells and these cells express Kell surface antigens. The expression of Kx antigen in progenitor cells was not determined owing to nonspecific reactions with the antibody. By cDNA blotting, mouse Kell expression was detected in bipotential megakaryocytes-erythroid cells and in colony-forming units-erythroid (CFU-E) and burst-forming units-erythroid (BFU-E), whereas XK was only detected in CFU-E and BFU-E. CONCLUSION: Both Kell and XK transcripts occur early during erythropoiesis; however, expression may not be coincident because, in mice, Kell transcripts, but not XK, occur in bipotential megakaryocytes-erythroid progenitor cells.

Amino Acid Transport Systems, Neutral↗
PubMed2003

Active amino acids of the Kell blood group protein and model of the ectodomain based on the structure of neutral endopeptidase 24.11.

In addition to its importance in transfusion, Kell protein is a member of the M13 family of zinc endopeptidases and functions as an endothelin-3-converting enzyme. To obtain information on the structure of Kell protein we built a model based on the crystal structure of the ectodomain of neutral endopeptidase 24.11 (NEP). Similar to NEP, the Kell protein has 2 globular domains consisting mostly of alpha-helical segments. The domain situated closest to the membrane contains both the N- and C-terminal sequences and the enzyme-active site. The outer domain contains all of the amino acids whose substitutions lead to different Kell blood group phenotypes. In the model, the zinc peptidase inhibitor, phosphoramidon, was docked in the active site. Site-directed mutagenesis of amino acids in the active site was performed and the enzymatic activities of expressed mutant Kell proteins analyzed and compared with NEP. Our studies indicate that Kell and NEP use the same homologous amino acids in the coordination of zinc and in peptide hydrolysis. However, Kell uses different amino acids than NEP in substrate binding and appears to have more flexibility in the composition of amino acids allowed in the active site.

Amino Acid Sequence↗
PubMed2003

Mutations that diminish expression of Kell surface protein and lead to the Kmod RBC phenotype.

BACKGROUND: Kmod is an inherited rare RBC phenotype characterized by weak but detectable expression of high-incidence Kell antigens. STUDY DESIGN AND METHODS: The 19 exons and the intron-exon regions of the KEL gene from four unrelated Kmod individuals were sequenced and compared to wild-type KEL. To study the mechanisms by which the mutations result in depression of Kell antigens, mutant and wild-type Kell proteins were expressed in 293T cells and the amounts of protein present on the cell surface were determined. RESULTS: The following point mutations were identified: Kmod-1, homozygous 1208G>A, S363N; Kmod-2, heterozygous, 1208G>A, S363N and 2150 A>G, Y677C; Kmod-3 (previously classified as KEL:-13), heterozygous 1106T>C, L329P and 1716G>A, W532Stop; Kmod-4, heterozygous, 2227G>A, G703R and a silent 1839C>T mutation. In transfected 293T cells, fewer G703R and L329P mutant Kell proteins were transported to the cell surface compared with wild-type Kell protein, and there was no detectable Y677C mutant Kell protein. Previously, it was shown that that S363N Kell protein was not detected on the cell surface. CONCLUSION: Different point mutations, causing amino acid substitutions and presumably altering protein conformation, inhibit transport of the mutant Kell proteins to the cell surface. The different mutations leading to the Kmod phenotype explain why anti-Ku made by persons with the Kmod phenotype are not mutually compatible.

Amino Acid Substitution↗
PubMed2003

Oxysterols suppress constitutive fibrinogen expression.

Elevated levels of both fibrinogen and cholesterol are risk factors in coronary artery disease. Previously we reported a metabolic link between fibrinogen and lipid metabolism in that HepG2 cells that were programmed by transfection of Bbeta-fibrinogen cDNA to overexpress fibrinogen exhibited increased synthesis of cholesterol and increased secretion of apolipoprotein B. In this study we demonstrate that oxysterols, which participate in maintaining cholesterol homeostasis, also down regulate fibrinogen expression. Treatment of HepG2 cells with 25-hydroxycholesterol lowered fibrinogen Aalpha, Bbeta and gamma mRNA levels and inhibited fibrinogen synthesis and secretion but had no effect on alpha1 -antitrypsin which, like fibrinogen, is an acute-phase protein. The inhibition of fibrinogen synthesis by oxysterols was maintained in interleukin-6 treated cells. Other oxysterols, that inhibit cholesterol synthesis by a feedback mechanism, also diminished fibrinogen expression in HepG2, rat H-4-II-E hepatoma cells and in primary human hepatocytes. Overexpression of SREBP-1 and SREBP-2 by transfection of HepG2 cells, or treatment with a synthetic LXRalpha agonist, which affect cholesterol metabolism, did not affect fibrinogen expression. We conclude that fibrinogen and cholesterol may share a novel common regulatory pathway.

Acute-Phase Reaction↗
PubMed2002

Point mutations causing the McLeod phenotype.

BACKGROUND: The McLeod phenotype is defined by absence of Kx, weakening of Kell system antigens, and acanthocytosis. Individuals with the McLeod phenotype usually develop late-onset neuromuscular abnormalities. Gene deletions, insertions, and point mutations that affect RNA splicing or that lead to premature stop codons have been reported to cause the McLeod phenotype. The McLeod phenotype may also be caused by mutations at a different splice site and by a novel mutation encoding an amino acid substitution that prevents transport to the cell surface. STUDY DESIGN AND METHODS: The coding and flanking intron regions of XK from four male, unrelated individuals with the McLeod phenotype and non-chronic granulomatous disease were sequenced and compared with the wild type sequence. Genomic DNA was amplified by PCR, and the products were sequenced. In one case, the mutant cDNA was expressed in a heterologous cell, and cell surface expression was determined. RESULTS: Three individuals with the McLeod phenotype had mutations that disrupted conserved GT sequences present at RNA splice sites. Two of them had G>C mutations at the 5' splice site of intron 1, and one had a G>A mutation at the 5' splice site of intron 2. One person with the McLeod phenotype had a 746C>G mutation in exon 3 encoding an R222G substitution. In a transfected cell, the expressed protein from the latter mutant did not travel to the cell surface. CONCLUSION: The McLeod phenotype may be caused by several different mutations.

Acanthocytes↗