ABSTRACTcDNA clones encoding the major subunit of the Duffy blood group were isolated from a human bone marrow cDNA library using a PCR-amplifled DNA anti-Duffy murine monoclonal antibody reacted with a synthetic peptide deduced from the cDNA clone. Hydropathy analysis suggested the presence of9 membrane-spanning a-helices. In bone marrow RNA blot analysis, the gpD cDNA detected.a 1.27-kb mRNA in Duffy-positive but not in Duffynegative individuals. It also identified the same size mRNA in adult kidney, adult spleen, and fetal liver; in brain, it detected a prominent 8.5-kb and a minor 2.2-kb mRNA. In Southern blot analysis, gpD cDNA identified a single gene in Duffypositive and -negative individuals. Duffy-negative individuals, therefore, have the gpD gene, but it is not expressed in bone marrow. The same or a similar gene is active in adult kidney, adult spleen, and fetal liver of Duffy-positive individuals. Whether this is true in Duffy-negative individuals remains to be demonstrated. A GenBank sequence search yielded a significant protein sequence homology to human and rabbit interleukin-8 receptors.
SummaryPlasmodium vivax and the related simian malarial parasite P. knowlesi use the DuffT blood group antigen as a receptor to invade human erythrocytes and region II of the parasite ligands for binding to this erythrocyte receptor. Here, we identify the peptide within the Duffy blood group antigen of human and rhesus erythrocytes to which the P. vivax and P. knowlesi ligands bind. Peptides from the NH2-terminal extracellular region of the Duffy antigen were tested for their ability to block the binding of erythrocytes to transfected Cos cells expressing on their surface region II of the Duffy-binding ligands. The binding site on the human Duffy antigen used by both the P. vivax and P. knowlesi ligands maps to a 35-amino acid region. A 34-amino acid peptide from the equivalent region of the rhesus Duffy antigen blocked the binding of P. vivax to human erythrocytes, although the P. vivax ligand expressed on Cos cells does not bind rhesus erythrocytes. The binding of the rhesus peptide, but not the rhesus erythrocyte, to the P. vivax ligand was explained by interference of carbohydrate with the binding process. Rhesus erythrocytes, treated with N-glycanase, bound specifically to P. vivax region II. Thus, the interaction of P. vivax ligand with human and rhesus erythrocytes appears to be mediated by a peptide-peptide interaction. Glycosylation of the rhesus Duffy antigen appears to block binding of the P. vivax ligand to rhesus erythrocytes.
Rat liver nuclei deprived of chromatin and nucleoplasm show a spongelike network which preserves its connection with nucleoli, the inner membrane of the nuclear envelope, and nuclear pore complexes. It contains all of the HnRNA, provided the endogenous proteolytic activity is inhibited by a proteolytic inhibitor such as phenylmethyl sulfonyl chloride (PMSC) or the fluoride form (PMSF). In the absence of these proteolytic inhibitors, HnRNA is dissociated from the spongelike network and sediments in a sucrose gradient as polydispersed ribonucleoprotein complexes. Furthermore, purified HnRNA as well as rRNA do not bind to the spongelike network when added to these nuclei. These observations demonstrate that the association of HnRNA to the nuclear skeleton is not an artifact. RNase treatment of the spongelike network digests the majority of the rapidly labeled RNA but does not alter the morphological aspect nor the architecture of this network. EDTA and heparin treatments affect neither the attachment of HnRNA nor the structural organization of this network. Electron microscope studies of the network reveal a characteristic flexuous configuration. Its relationship with diffused and condensed chromatin is discussed. KEY WORDS nuclear skeleton proteases 9 ribonucleoprotein complexes 9 heterogeneous RNA nudeal" From our studies on rat liver and ascites tumor nuclei, evidence has been obtained that ribonucleoprotein (RNP) complexes containing rapidly labeled RNA (HnRNA and presumably mRNA) are part of an RNP-network which in turn appears to be tightly bound to the nuclear envelope (11). Dissolution of this envelope by nonionic detergent doe not release labeled RNA (11). Aaronson and Blobel (1) and Scheer and co-workers (32) have found that similar treatment removes lipids from the nuclear envelope and that such membrane-denudated nuclei retain both their shape and pore complexes. They reported that these nuclear pore complexes are attached to intranuclear structures, particularly to a distinct peripheral layer of ill-defined nuclear material which they have interpreted as equivalent to the fibrous lamina described in a variety of cell types (14,30,33,35). Berezney and Coffey have shown that a residual nuclear structure which preserves the nuclear shape remains after rat liver nuclei are treated with high salt and digested with ribonucle-
All of the antigenic determinants of the Duffy blood group system are in a glycoprotein (gp-Fy), which is encoded by a single-copy gene (FY) located on chromosome 1. gp-Fy is also produced in several cell types, including endothelial cells of capillary and postcapillary venules, the epithelial cell of kidney collecting ducts, lung alveoli, and the Purkinje cells of the cerebellum. This protein, which spans the cell membrane seven times, is a member of the superfamily of chemokine receptors and a malarial parasite receptor. The mouse Duffy gene (Dfy) homolog of human FY is also a single-copy gene, which maps in a region of conserved synteny with FY and produces a glycoprotein with 60% homology to the human protein. The mouse Duffy-like protein also binds chemokines. To study the biological role of gp-Fy, we generated a mouse strain in which Dfy was deleted. These homozygous Dfy ؊/؊ mice were indistinguishable in size, development, and health from wild-type and heterozygous littermates. We also examined components of the immune system and found no differences in lymph nodes or peripheral blood leukocyte levels between knockout and wild-type mice. The gross and histological anatomy of the thymus, spleen, lung, and brain showed no significant differences between mutants and wild-type mice. There was no indication of an overall difference between the knockout and wild-type mice in systematic neurological examinations. The only significant difference between Dfy ؊/؊ and Dfy ؉/؉ mice that we found was in neutrophil migration in peritoneal inflammations induced by lipopolysaccharide and thioglycolate. In mice homozygous for the deletion, there was less neutrophil recruitment into the peritoneal cavity and neutrophil influx in the intestines and lungs than in wild-type mice. Despite this, the susceptibility to Staphylococcus aureus infection was the same in the absence and in the presence of gp-Fy. Our results indicate that gp-Fy is functionally a redundant protein that may participate in the neutrophil migratory process.
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