Colvin M. Redman

Association of XK and Kell blood group proteins.

A disulfide bond links Kell and XK red cell membrane proteins. Kell, a type II membrane glycoprotein, carries over 20 blood group antigens, and XK, which spans the membrane 10 times, is lacking in rare individuals with the McLeod syndrome. Kell is classified in the neprilysin family of zinc endopeptidases, and XK has structural features that suggest it is a transport protein. Kell has 15 extracellular cysteines, and XK has one in its fifth extracellular loop. Five of the extracellular cysteine residues in Kell are not conserved in the other members of the neprilysin family, and based on the hypothesis that one of the nonconserved cysteines is linked to XK, cysteines 72 and 319 were mutated to serine. The single extracellular cysteine 347 of XK was also mutated. Co-expression of combinations of wild-type and mutant proteins in transfected COS-1 cells showed that Kell C72S did not form a Kell-XK complex with wild-type XK, while wild-type Kell and Kell C319S did. XK C347S was also unable to form a complex with wild-type Kell, indicating that Kell cysteine 72 is linked to XK cysteine 347. Kell C72S was transported to the cell surface, indicating that linkage to XK is not required. In addition, chemical cross-linking of red cell membranes with dithiobispropionimidate indicated that glyceraldehyde-3-phosphate dehydrogenase is a near neighbor of Kell.

The Kell Blood Group System: Kell and XK Membrane Proteins

Two membrane proteins express the antigens that comprise the Kell blood group system. A single antigen, Kx, is carried on XK, a 440-amino acid protein that spans the membrane 10 times, and more than 20 antigens reside on Kell, a 93-kd, type II glycoprotein. XK and Kell are linked, close to the membrane surface, by a single disulfide bond between Kell cysteine 72 and XK cysteine 347. Although primarily expressed in erythroid tissues, Kell and XK are also present in many other tissues. The polymorphic forms of Kell are due to single base mutations that encode different amino acids. Some Kell antigens are highly immunogenic and may cause strong reactions if mismatched blood is transfused and severe fetal anemia in sensitized mothers. Antibodies to KEL1 may suppress erythropoiesis at the progenitor level, leading to fetal anemia. The cellular functions of Kell/XK are complex. Absence of XK, the McLeod phenotype, is associated with acanthocytic red blood cells (RBCs), and with late-onset forms of muscular dystrophy and nerve abnormalities. Kell, by homology, is a member of the neprilysin (M13) family of membrane zinc endopeptidases and it preferentially activates endothelin-3 by specific cleavage of the Trp21-Ile22 bond of big endothelin-3.

Functional and structural aspects of the Kell blood group system.

Two covalently linked proteins, Kell and XK, constitute the Kell blood group system. Kell, a 93-Kd type II glycoprotein, is highly polymorphic and carries all but 1 of the known Kell antigens, and XK, which traverses the membrane 10 times, carries a single antigen, the ubiquitous Kx. The Kell/XK complex is not limited to erythroid tissues and may have multiple physiological roles. Absence of one of the component proteins, XK, is associated with abnormal red cell morphology and late-onset forms of nerve and muscle abnormalities, whereas the other protein component, Kell, is an enzyme whose principal known function is the production of a potent bioactive peptide, ET-3.

Kell and XK immunohistochemistry in McLeod myopathy

The McLeod syndrome is an X‐linked neuroacanthocytosis manifesting with myopathy and progressive chorea. It is caused by mutations of the XK gene encoding the XK protein, a putative membrane transport protein of yet unknown function. In erythroid tissues, XK forms a functional complex with the Kell glycoprotein. Here, we present an immunohistochemical study in skeletal muscle of normal controls and a McLeod patient with a XK gene point mutation (C977T) using affinity‐purified antibodies against XK and Kell proteins. Histological examination of the affected muscle revealed the typical pattern of McLeod myopathy including type 2 fiber atrophy. In control muscles, Kell immunohistochemistry stained sarcoplasmic membranes. XK immunohistochemistry resulted in a type 2 fiber‐specific intracellular staining that was most probably confined to the sarcoplasmic reticulum. In contrast, there was only a weak background signal without a specific staining pattern for XK and Kell in the McLeod muscle. Our results demonstrate that the lack of physiological XK expression correlates to the type 2 fiber atrophy in McLeod myopathy, and suggest that the XK protein represents a crucial factor for the maintenance of normal muscle structure and function.

The intracellular pathway of newly formed rat liver catalase

Abstract [1- 14 C]- l -leucine was injected into the femoral vein of male rats. At various times after injection, from 3 to 120 min, the livers were removed and fractionated into various cell components. The intracellular location of radioactive catalase and albumin were compared. Radioactive catalase and albumin both appeared at 5–10 min after injection but in different intracellular locations. Catalase was first located in the soluble cytoplasmic fraction while albumin was found in the rough endoplasmic reticulum. Peak catalase radioactivity did not occur in the rough endoplasmic reticulum until 20–40 min after injection, at which time albumin was already leaving the smooth endoplasmic reticulum. Radioactive catalase did not enter the smooth endoplasmic reticulum. The catalase which sedimented with the rough endoplasmic reticulum could be separated by sucrose gradient centrifugation from microsomal vesicles which contained nascent albumin. The radioactive catalase accumulated with time into a peroxisomal fraction but some catalase of equal specific radioactivity remained in the soluble cytoplasmic fraction even at 120 min after injection.

Fibrinogen Assembly and Secretion ROLE OF INTRACHAIN DISULFIDE LOOPS

Human fibrinogen is a homodimer composed of three different (Aα, Bβ, γ) polypeptide chains. The chains are linked by 29 inter- and intrachain disulfide bonds. Each half-molecule has 6 intrachain disulfide bonds, which form loops in the carboxyl-terminal region of each of the chains. Aα chain has one disufide loop (Cys442-Cys472), Bβ has three (Cys201-Cys286, Cys211-Cys240, and Cys394-Cys407), and γ has two loops (Cys153-Cys182 and Cys326-Cys339). The intrachain loops are conserved in fibrinogens of different species. We changed, by site-directed mutagenesis, the cysteines, which form the intrachain loops, to serine or alanine. Fibrinogen chain assembly and secretion was determined in transiently transfected COS cells expressing two normal and a mutant fibrinogen chain. In the Bβ and γ chains, disruption of the disulfide loops closest to the “coiled-coil” region (CysBβ211-Cys240, CysBβ201-Cys286, and Cysγ153-Cys182) abolished chain assembly and secretion, indicating that the disulfide loops closest to the coiled-coil region are essential for chain assembly. By contrast, preventing formation of the disulfide loops, which are toward the carboxyl termini of each of the chains, had different effects. Disruption of the single Aα disulfide loop had no effect, as did disruption of BβCys394-Cys407. However, disruption of Cysγ326-Cys339, which is similar in size and location to CysBβ394-Cys407, allowed chain assembly to occur, but the assembled chains were not secreted.

Localization of enzymes involve in polyphosphoinositide metabolism on the cytoplasmic surface of the human erythrocyte membrane

Abstract 1. 1. Impermeable inside-out and right-side-out vesicles were prepared from membranes of human erythrocytes. During preparation of each kind of impermeable vesicle, permeable vesicles were also obtained. 2. 2. Incubation of vesicles with [γ-32P]ATP at 37 °C for periods of up to 1 h did not change the topography or the permeability of the vesicles. 3. 3. Vesicles incorporated labeled phosphate from [γ-32P]ATP into both diphosphoinositide and triphosphoinositide, but impermeable inside-out vesicles incorporated significantly more nuclide than did right-side-out vesicles. 4. 4. Permeable vesicles derived during the preparation of inside-out vesicles were as active as impermeable inside-out vesicles in the incorporation of labeled phosphate into the polyphosphoinositides. However, permeable vesicles derived during the preparation of right-side-out vesicles were not as active. 5. 5. Impermeable right-side-out vesicles, treated with 0.01% saponin, incorporated labeled phosphate into the polyphosphoinositides at a level comparable to that of impermeable inside-out vesicles. 6. 6. These data show that the enzymes involved in metabolism of diphosphoinositide and triphosphoinositide are located on the cytoplasmic surface of the erythrocyte membrane.

Molecular basis of the K:6,-7 [Js(a+b−)] phenotype in the Kell blood group system

BACKGROUND: The Kell blood group system consists of at least 21 antigens. KEL6(Jsa) is a low‐incidence antigen that has an antithetical relationship with the high‐incidence KEL7(Jsb) antigen. The molecular basis of KEL6 that appears in less than 1.0 percent of the general population, but in up to 19.5 percent of African Americans, was unknown.

Secretion of Biologically Active Recombinant Fibrinogen by Yeast

Fibrinogen (340 kDa) is a plasma protein that plays an important role in the final stages of blood clotting. Human fibrinogen is a dimer with each half-molecule composed of three different polypeptides (Aα, 67 kDa; Bβ, 57 kDa; , 47 kDa). To understand the mechanism of fibrinogen chain assembly and secretion and to obtain a system capable of producing substantial amounts of fibrinogen for structure-function studies, we developed a recombinant system capable of secreting fibrinogen. An expression vector (pYES2) was constructed with individual fibrinogen chain cDNAs under the control of a Gal-1 promoter fused with mating factor Fα1 prepro secretion signal (SS) cascade. In addition, other constructs were prepared with combinations of cDNAs encoding two chains or all three chains in tandem. Each chain was under the control of the Gal-1 promoter. These constructs were used to transform Saccharomyces cerevisiae (INVSC1; Matα his3-Δ1 leu2 trp1-289 ura3-52) in selective media. Single colonies from transformed yeast cells were grown in synthetic media with 4% raffinose to a density of 1 × 108 cells/ml and induced with 2% galactose for 16 h. Yeast cells expressing all three chains contained fibrinogen precursors and nascent fibrinogen and secreted about 30 μg/ml of fibrinogen into the culture medium. The Bβ and chains, but not Aα, were glycosylated. Glycosylation of Bβ and chains was inhibited by treatment of transformed yeast cells with tunicamycin. Intracellular Bβ and chains, but not the Aα chains in secreted fibrinogen, were cleaved by endoglycosidase H. Carbohydrate analysis indicated that secreted recombinant fibrinogen contained N-linked asialo-galactosylated biantennary oligosaccharide. Recombinant fibrinogen yielded the characteristic plasmin digestion products, fragments D and E, that were immunologically indistinct from the same fragments obtained from plasma fibrinogen. The recombinant fibrinogen was shown to be biologically active in that it could form a thrombin-induced clot, which, in the presence of factor XIIIa, could undergo chain dimerization and Aα chain polymer formation.

Isolation of Kell‐active protein from the red cell membrane

Kell blood‐group‐active protein has been isolated by labeling red cell surface proteins with 125I, sensitizing intact cells with anti‐K1, anti‐ K2, anti‐K7, or anti‐K22, solubilizing the cell membranes, isolating immune complexes, and separating their components by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Each antibody separated a protein of approximately 93,000 daltons. Periodic‐acid Schiff (PAS) staining of Kell protein showed that it was glycosylated. When separated under non‐reducing conditions, Kell protein had different SDS‐PAGE characteristics with protein bands of approximately 85,000 daltons and 115,000 daltons. This suggests that in the red cell membrane Kell protein is complexed with other proteins. Quantitative experiments made with anti‐K7, anti‐K22, and a mixture of anti‐K7 and anti‐K22 indicate that both antigen specificities are present in the same molecule. These biochemical data support serological studies which indicate that K22 is part of the Kell system.