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Molecular cloning of a glycosylphosphatidylinositolanchored molecule CDw108
246 same family, and share 36% identity. Despite their importance in transfusion medicine, the function of these proteins, individually or together, has not been established. Marini et al have previously shown sequence similarity between the Rh family proteins and Mep/Amt ammonium transporters, and in the current paper, they show that when RhAG is expressed in Mep mutant yeast cells, Rh50 transports ammonia. They obtained similar results with RhGK (also called RhCG by Dr Huang's group), which is a new human homologue expressed in kidney cells. RhAG and RhGK specifically corrected the growth defect of the yeast mutant deficient in ammonium uptake. Ammonium efflux assays and growth tests in the presence of toxic concentrations of the analogue methylammonium indicate that RhAG and RhGK also promote ammonium export. This provides experimental evidence for a role of Rh50 in ammonium transport, of which, in humans, little is known. (M.E.R.)
Expression of Kell blood group protein in nonerythroid tissues. D. Russo, X. Wu, C.M. Redman, et al. Blood 96:340-346, 2000. The Kell blood group protein is a zinc endopeptidase that cleaves big endothelin-3 (an intermediate precursor) to yield endothelin-3, which is a potent bioactive peptide. In red blood cells, the Kell blood group protein is linked by a disulfide bond to the XK protein. Previous studies indicated that Kell protein is primarily expressed in erythroid tissues, whereas XK protein has a wide tissue distribution. In this paper, the authors confirmed by Northern blot analysis and PCR-screening of tissue complementary DNAs (cDNAs) that Kell protein is primarily expressed in erythroid tissues and show that it is also expressed in a nearly equal amount in testes and that it has weaker expression in a number of other tissues. Sufficient Kell protein is present in testes and skeletal muscle to be detected by Western immunoblot analysis. The studies described here showed that Kell protein is also disulfide linked to XK protein in nonerythroid tissues, thereby indicating possible complementary functions for XK and Kell proteins. Kell cDNA obtained by PCR amplification from skeletal muscle had an identical sequence to that obtained from erythroid tissues, with the possible exception of a nncleotide substitution (awaiting confirmation) resulting in a predicted amino acid substitution (Asp692Asn). This suggests that erythroid and nonerythroid forms of Kell protein have shnilar enzymatic functions. (M.E.R.)
Structure of human neutral endopeptidase (Neprilysin) complexed with phosphoramidon. C. Oefner, A. D'Arcy, M. Hennig, et al. J Mol Biol 296:341-349, 2000. Neprilysin (NEP, CALLA, CD10) is the prototype of a group of metallopeptidases that includes the endothelin-converting enzymes (ECE-1 and ECE-2), the PEX gene product, and the Kell protein. These proteins belong to the MI3 subfamily of mammalian neutral endopeptidases and consist of a short Ntenninal cytoplasmic domain, a single trans-membrane helix, and a large C-terminal extracellular domain that contains the active site. The crystal structure of the extracellular domain (amino acid residues 52 to 749) of human NEP complexed with the generic metalloproteinase inhibitor, phosphoramidon, is described. The structure shows 2 multiply connectedfolding domains, which embrace a large central cavity that contains the active site. The inhibitor is bound to 1 side of the cavity, and its
JOURNAL CLUB binding mode provides a detailed understanding of the ligand binding. It is possible that understanding the structure of this homologous protein may shed light on the 3-dimensional structure of the Kell protein. (M.E.R.)
Molecular heterogeneity of the Jknu, phenotype: Expression analysis of the Jk ($291P) mutation found in Finns. F. Sidoux-Wa/ter, N. Lucien, R. Nissinen, eta/. Blood 96:1566-1573, 2000. The Jknull phenotype lacks antigens in the Kidd blood group system and the Kidd protein (the urea transporter in red ceils). The molecular basis underlying the J k ( a - b - ) phenotype found in Polynesians is a g > a mutation in the 3'-acceptor splice site in intron 5. This results in a deletion of exon 6. In Finns, the J k ( a - b - ) phenotype is because of a T > C substitution resulting in a Ser291Pro change in the consensus N-glycosylation site. Expression studies in Xenopus oocytes showed that the Jk(S291P) protein functions as a urea transporter, but the transport activity and the membrane expression Ievel of the mutant protein were reduced. A substantial fraction of the mutant protein was retained intracellularly, suggesting that the transit to the plasma membrane was reduced. Although the wild-type protein was expressed at the cell surface of transfected K562 cells, the $291P mutant was not. The $291P mutant protein is not present in human RBCs. The authors conclude that this single amino acid substitution is responsible for the J k ( a - b - ) phenotype in Finns. These results were obtained by testing 32 unrelated Jk(a b - ) people. The 14 Polynesians were Samoan, Niuean, Tongan, and Hawaiian. The g > a 3'-acceptor splice site mutation was also found in 1 person each from the United States, Australia, and Europe and from 1 Chinese (previously reported and not counted in the 32 probands reported here). The $291P mutation was found in 15 Finns. A third mutation underlies the J k ( a - b ) phenotype (described previously) in a French proband. In this person, a mutation in the invariant g residue of the 5"-donor splice site of intron 7 causes deletion of exon 7 and no Kidd protein in the red cell membrane. Based on these findings, it would be wise to perform analysis for the g > a mutation in exon 5 and the $291P mutation when performing the Jk genotyping in Polynesians and Finns. (M.E.R.)
Molecular cloning of a glycosylphosphatidylinositolanchored molecule CDwl08. A. Yamada, K. Kubo, T. Takeshita, et al. J Immunol 162:4094-4100, 1999. CD108 encodes an 80-kDa glycosylphosphatidylinositol (GPI)-liuked membrane glycoprotein, which has been shown previously to encode the JMH blood group. Yamada et al describe cloning 1998 base pairs of cDNA encoding a protein of 666 amino acids (aa), including a signal peptide (46aa) and a GPI-anchor motif (19aa). Thus, the mature CD108 protein consists of 602aa and has an estimated mass (without carbohydrates) of 68 kDa, the RGD (Arg-Gly-Asp) cell attachment sequence and 5 potential N-glycosylation sites. CDI08 mRNA is expressed in peripheral blood mononuclear cells, spleen, thymus, testis, placenta, and brain but not in prostate, uterus, small intestine, colon, heart, lung, liver, skeletal muscle, kidney, or pancreas. The CDI08 gene is located on the long arm of chromosome 15 at 15q23-24. Because high incidence antigen JMH was known to be carried on CDI08 and the gene that encodes CD108 has now been cloned, the JMH antigen was
JOURNAL CLUB promoted to blood group system status in July 2000 by the International Society of Blood Transfusion Working Party on Terminology for Red Cell Surface Antigens. (M.E.R.)
Identification of the Dombrock blood group glycoprotein as a polymorphic member of the ADP-ribosyltransferase gene family. A. Gubin, J,M. Njoroge, U. Wojda, et a/. Blood 96:2621-2627, 2000. This paper describes the cloning of the Dombrock gene using a novel approach. Dombrock is expressed on erythroid cells by a GPI-anchor and its gene is located at chromosomal location 12p. Based on this knowledge, in silico analyses of nearly 5000 expressed sequence tags (ESTs) from terminally differentiating human erytfiroid cells were performed. A candidate gene was identified and transfection experiments showed specific binding of anti-Dombrock and confirmed glycosylphosphatidyl-inositol membrane attachment. Dombrock is expressed in human spleen, lymph node, thymus, peripheral blood leukocytes, bone marrow, and fetal liver. Homology studies suggest that the Dombrock glycoprotein is a member of the adenosine 5'diphosphate (ADP)-ribosyltransferase ectoenzyme gene family. However, enzyme activity of Dombrock has not been shown in red cells. Sequence analysis of samples from known Do ( A + B - ) and Do (A B + ) donors showed a single amino acid substitution (Asn265Asp) within the RGD motif of the molecule. Two other single nucleotide polymorphisms distinguish DOA from DOB, but both are silent at the amino acid level. With the cloning of Dombrock, genes encoding only 3 blood group systems (SC, P1, RAPH) await to be cloned.
(M.E.R.) Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. E. Lagasse, H. Connors, M. AI-Dhalirny, et al. Nature Medicine 6:1229-1234, 2000. This group not only confirmed that bone marrow could generate hepatocytes but found that purified hematopoietic stem cells (HSCs) alone could generate functional hepatocytes. The authors used lethal irradiation in female F A H - / F A H - mice, which were subsequently rescued with bone marrow. FAH / F A H - mice are a model of fatal hereditary tyrosinemia type I, which must be treated with 2-(2-nitro-4-trifluoro-methyIbenzyol)-l,3-cyclohexanedione (NTBC), which bypasses the enzyme deficiency, The mice were transplanted with unfractionated bone man'ow from male, wild-type mice transgenic for Escherichia coli LacZ. After discontinuing treatment with NTBC, the recipients were found to have substantial liver repopulation with male donor cells as docmnented by Y chromosome in situ hybridization (ISH) and/3-galactosidase staining of up to 50%. Functionality was close to that of normal controls. Next, the authors purified hematopoietic stem cells by fluorescence activated cells sorting (FACS). The ceils selected were c-kithigt~, Thy l~ Lin-, and Sca-1 + and were called KTLS cells. (Lin includes several antigens, all of which suggest specific lineage evolution, eg, B-cell, T-cell, and so on.) These cells were then confirmed to be CD45+ to ensure that they were hematopoietic cells. After this rigorous selection process, these cells were infused intravenously at very low numbers (10, 50, 100, or 1000) into women. In addition to these celis, the mice also received marrow cells from F A H - / F A H - women to
247 ensure survival after lethal irradiation. The transplanted mice were maintained on NTBC for 2 months after which time NTBC was withdrawn until the mice were noted to drop 30% of their body weight and NTBC was restarted. NTBC withdrawal was repeated once, and then the mice were sacrificed for evaluation. The mice were noted to have long-term marrow engraftment of at least 10% cells derived from the male HSCs in those mice transplanted with 50, 100, or 1000 HSCs but not with 10 HSCs. The liver was analyzed using/3-galactosidase histochemical staining, FAH immunohistochemistry, and Y chromosome ISH. The histologic findings indicated that nodules positive for all 3 parameters evaluated were present confirming the ability of KTLS HSCs to functionally repopulate the damaged liver in this disease state. A converse study in which KTLS cells were mostly omitted from the transpIant showed that they were essential to significantly repopulate the damaged liver. Findings from these experiments show therapeutic benefit in a well-defined disease state and suggest that these maneuvers may provide benefit in humans. Stem cells that seemingly cross classic germ lines are of substantial interest. This, and other similar studies, may show previously unexplored territory for bone marrow transplant therapies. (C.H.)
Transplantation of bone marrow as compared with peripheral-blood cells from HLA-identical relatives In patients with hematologic cancers. W./. Bensinger, P.J. Martin, B. Storer, et al. NEJM 344:175-181, 2001. This multicenter, randomized trial was conducted to detect differences in acute graft-versus-host-disease (GVHD) stages II, IH, and IV between peripheral blood stem cell transplant (PBSCT) and standard bone marrow transplant (BMT) given to patients with cancers of hematologic origin. Secondary end points were also recorded, including time to engraftment for neutrophils and platelets, overall survival, and disease-free survival, with some interesting results. Ultimately, 172 patients received related HLA identical products derived from either peripheral blood stem cells (n = 81) of at least 5 • l0 s CD34+ cells/kg from donors mobilized with 16 /,g/kg subcutaneous G-CSF for 5 days or bone marrow (n = 91) collected by standard techniques. Neutrophil recovery was found to occur 5 days (at day 16) earlier in those patients given PBSCTs, and similarly platelets recovered 6 days (at day 13) earlier. Neutrophil recovery was defined as exceeding a neutrophil count > 500/mm 3 and platelet recovery was defined as exceeding a count greater than 20,000/mm 3. No statistically significant differences were detected in the incidence of GVHD grades II, III, or IV (64% PBSCT v 57% BMT), nor were there any statistically significant differences in the occurrence of chronic GVHD (46% and 35%, respectively). There was no difference in death at 2 years between PBSCT and BMT (21% and 30 %, respectively, [P = .24]), whereas the incidence of relapse at 2 years was i4% and 25%, respectively, (P = .04). For overall and disease-free survival at 2 years, the percentages were 66% and 54% (PBSCT and BMT, P - .06) and 65% and 45% (PBSCT and BMT, P = .03). In the subgroups of patients with less advanced versus more advanced cancers, there was no difference between PBSCT and BMT in those with iess severe disease for 2-year survival. For those with more advanced disease, however, there was a 57% survival rate
Expression of Kell blood group protein in nonerythroid tissues. D. Russo, X. Wu, C.M. Redman, et al. Blood 96:340-346, 2000. The Kell blood group protein is a zinc endopeptidase that cleaves big endothelin-3 (an intermediate precursor) to yield endothelin-3, which is a potent bioactive peptide. In red blood cells, the Kell blood group protein is linked by a disulfide bond to the XK protein. Previous studies indicated that Kell protein is primarily expressed in erythroid tissues, whereas XK protein has a wide tissue distribution. In this paper, the authors confirmed by Northern blot analysis and PCR-screening of tissue complementary DNAs (cDNAs) that Kell protein is primarily expressed in erythroid tissues and show that it is also expressed in a nearly equal amount in testes and that it has weaker expression in a number of other tissues. Sufficient Kell protein is present in testes and skeletal muscle to be detected by Western immunoblot analysis. The studies described here showed that Kell protein is also disulfide linked to XK protein in nonerythroid tissues, thereby indicating possible complementary functions for XK and Kell proteins. Kell cDNA obtained by PCR amplification from skeletal muscle had an identical sequence to that obtained from erythroid tissues, with the possible exception of a nncleotide substitution (awaiting confirmation) resulting in a predicted amino acid substitution (Asp692Asn). This suggests that erythroid and nonerythroid forms of Kell protein have shnilar enzymatic functions. (M.E.R.)
Structure of human neutral endopeptidase (Neprilysin) complexed with phosphoramidon. C. Oefner, A. D'Arcy, M. Hennig, et al. J Mol Biol 296:341-349, 2000. Neprilysin (NEP, CALLA, CD10) is the prototype of a group of metallopeptidases that includes the endothelin-converting enzymes (ECE-1 and ECE-2), the PEX gene product, and the Kell protein. These proteins belong to the MI3 subfamily of mammalian neutral endopeptidases and consist of a short Ntenninal cytoplasmic domain, a single trans-membrane helix, and a large C-terminal extracellular domain that contains the active site. The crystal structure of the extracellular domain (amino acid residues 52 to 749) of human NEP complexed with the generic metalloproteinase inhibitor, phosphoramidon, is described. The structure shows 2 multiply connectedfolding domains, which embrace a large central cavity that contains the active site. The inhibitor is bound to 1 side of the cavity, and its
JOURNAL CLUB binding mode provides a detailed understanding of the ligand binding. It is possible that understanding the structure of this homologous protein may shed light on the 3-dimensional structure of the Kell protein. (M.E.R.)
Molecular heterogeneity of the Jknu, phenotype: Expression analysis of the Jk ($291P) mutation found in Finns. F. Sidoux-Wa/ter, N. Lucien, R. Nissinen, eta/. Blood 96:1566-1573, 2000. The Jknull phenotype lacks antigens in the Kidd blood group system and the Kidd protein (the urea transporter in red ceils). The molecular basis underlying the J k ( a - b - ) phenotype found in Polynesians is a g > a mutation in the 3'-acceptor splice site in intron 5. This results in a deletion of exon 6. In Finns, the J k ( a - b - ) phenotype is because of a T > C substitution resulting in a Ser291Pro change in the consensus N-glycosylation site. Expression studies in Xenopus oocytes showed that the Jk(S291P) protein functions as a urea transporter, but the transport activity and the membrane expression Ievel of the mutant protein were reduced. A substantial fraction of the mutant protein was retained intracellularly, suggesting that the transit to the plasma membrane was reduced. Although the wild-type protein was expressed at the cell surface of transfected K562 cells, the $291P mutant was not. The $291P mutant protein is not present in human RBCs. The authors conclude that this single amino acid substitution is responsible for the J k ( a - b - ) phenotype in Finns. These results were obtained by testing 32 unrelated Jk(a b - ) people. The 14 Polynesians were Samoan, Niuean, Tongan, and Hawaiian. The g > a 3'-acceptor splice site mutation was also found in 1 person each from the United States, Australia, and Europe and from 1 Chinese (previously reported and not counted in the 32 probands reported here). The $291P mutation was found in 15 Finns. A third mutation underlies the J k ( a - b ) phenotype (described previously) in a French proband. In this person, a mutation in the invariant g residue of the 5"-donor splice site of intron 7 causes deletion of exon 7 and no Kidd protein in the red cell membrane. Based on these findings, it would be wise to perform analysis for the g > a mutation in exon 5 and the $291P mutation when performing the Jk genotyping in Polynesians and Finns. (M.E.R.)
Molecular cloning of a glycosylphosphatidylinositolanchored molecule CDwl08. A. Yamada, K. Kubo, T. Takeshita, et al. J Immunol 162:4094-4100, 1999. CD108 encodes an 80-kDa glycosylphosphatidylinositol (GPI)-liuked membrane glycoprotein, which has been shown previously to encode the JMH blood group. Yamada et al describe cloning 1998 base pairs of cDNA encoding a protein of 666 amino acids (aa), including a signal peptide (46aa) and a GPI-anchor motif (19aa). Thus, the mature CD108 protein consists of 602aa and has an estimated mass (without carbohydrates) of 68 kDa, the RGD (Arg-Gly-Asp) cell attachment sequence and 5 potential N-glycosylation sites. CDI08 mRNA is expressed in peripheral blood mononuclear cells, spleen, thymus, testis, placenta, and brain but not in prostate, uterus, small intestine, colon, heart, lung, liver, skeletal muscle, kidney, or pancreas. The CDI08 gene is located on the long arm of chromosome 15 at 15q23-24. Because high incidence antigen JMH was known to be carried on CDI08 and the gene that encodes CD108 has now been cloned, the JMH antigen was
JOURNAL CLUB promoted to blood group system status in July 2000 by the International Society of Blood Transfusion Working Party on Terminology for Red Cell Surface Antigens. (M.E.R.)
Identification of the Dombrock blood group glycoprotein as a polymorphic member of the ADP-ribosyltransferase gene family. A. Gubin, J,M. Njoroge, U. Wojda, et a/. Blood 96:2621-2627, 2000. This paper describes the cloning of the Dombrock gene using a novel approach. Dombrock is expressed on erythroid cells by a GPI-anchor and its gene is located at chromosomal location 12p. Based on this knowledge, in silico analyses of nearly 5000 expressed sequence tags (ESTs) from terminally differentiating human erytfiroid cells were performed. A candidate gene was identified and transfection experiments showed specific binding of anti-Dombrock and confirmed glycosylphosphatidyl-inositol membrane attachment. Dombrock is expressed in human spleen, lymph node, thymus, peripheral blood leukocytes, bone marrow, and fetal liver. Homology studies suggest that the Dombrock glycoprotein is a member of the adenosine 5'diphosphate (ADP)-ribosyltransferase ectoenzyme gene family. However, enzyme activity of Dombrock has not been shown in red cells. Sequence analysis of samples from known Do ( A + B - ) and Do (A B + ) donors showed a single amino acid substitution (Asn265Asp) within the RGD motif of the molecule. Two other single nucleotide polymorphisms distinguish DOA from DOB, but both are silent at the amino acid level. With the cloning of Dombrock, genes encoding only 3 blood group systems (SC, P1, RAPH) await to be cloned.
(M.E.R.) Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. E. Lagasse, H. Connors, M. AI-Dhalirny, et al. Nature Medicine 6:1229-1234, 2000. This group not only confirmed that bone marrow could generate hepatocytes but found that purified hematopoietic stem cells (HSCs) alone could generate functional hepatocytes. The authors used lethal irradiation in female F A H - / F A H - mice, which were subsequently rescued with bone marrow. FAH / F A H - mice are a model of fatal hereditary tyrosinemia type I, which must be treated with 2-(2-nitro-4-trifluoro-methyIbenzyol)-l,3-cyclohexanedione (NTBC), which bypasses the enzyme deficiency, The mice were transplanted with unfractionated bone man'ow from male, wild-type mice transgenic for Escherichia coli LacZ. After discontinuing treatment with NTBC, the recipients were found to have substantial liver repopulation with male donor cells as docmnented by Y chromosome in situ hybridization (ISH) and/3-galactosidase staining of up to 50%. Functionality was close to that of normal controls. Next, the authors purified hematopoietic stem cells by fluorescence activated cells sorting (FACS). The ceils selected were c-kithigt~, Thy l~ Lin-, and Sca-1 + and were called KTLS cells. (Lin includes several antigens, all of which suggest specific lineage evolution, eg, B-cell, T-cell, and so on.) These cells were then confirmed to be CD45+ to ensure that they were hematopoietic cells. After this rigorous selection process, these cells were infused intravenously at very low numbers (10, 50, 100, or 1000) into women. In addition to these celis, the mice also received marrow cells from F A H - / F A H - women to
247 ensure survival after lethal irradiation. The transplanted mice were maintained on NTBC for 2 months after which time NTBC was withdrawn until the mice were noted to drop 30% of their body weight and NTBC was restarted. NTBC withdrawal was repeated once, and then the mice were sacrificed for evaluation. The mice were noted to have long-term marrow engraftment of at least 10% cells derived from the male HSCs in those mice transplanted with 50, 100, or 1000 HSCs but not with 10 HSCs. The liver was analyzed using/3-galactosidase histochemical staining, FAH immunohistochemistry, and Y chromosome ISH. The histologic findings indicated that nodules positive for all 3 parameters evaluated were present confirming the ability of KTLS HSCs to functionally repopulate the damaged liver in this disease state. A converse study in which KTLS cells were mostly omitted from the transpIant showed that they were essential to significantly repopulate the damaged liver. Findings from these experiments show therapeutic benefit in a well-defined disease state and suggest that these maneuvers may provide benefit in humans. Stem cells that seemingly cross classic germ lines are of substantial interest. This, and other similar studies, may show previously unexplored territory for bone marrow transplant therapies. (C.H.)
Transplantation of bone marrow as compared with peripheral-blood cells from HLA-identical relatives In patients with hematologic cancers. W./. Bensinger, P.J. Martin, B. Storer, et al. NEJM 344:175-181, 2001. This multicenter, randomized trial was conducted to detect differences in acute graft-versus-host-disease (GVHD) stages II, IH, and IV between peripheral blood stem cell transplant (PBSCT) and standard bone marrow transplant (BMT) given to patients with cancers of hematologic origin. Secondary end points were also recorded, including time to engraftment for neutrophils and platelets, overall survival, and disease-free survival, with some interesting results. Ultimately, 172 patients received related HLA identical products derived from either peripheral blood stem cells (n = 81) of at least 5 • l0 s CD34+ cells/kg from donors mobilized with 16 /,g/kg subcutaneous G-CSF for 5 days or bone marrow (n = 91) collected by standard techniques. Neutrophil recovery was found to occur 5 days (at day 16) earlier in those patients given PBSCTs, and similarly platelets recovered 6 days (at day 13) earlier. Neutrophil recovery was defined as exceeding a neutrophil count > 500/mm 3 and platelet recovery was defined as exceeding a count greater than 20,000/mm 3. No statistically significant differences were detected in the incidence of GVHD grades II, III, or IV (64% PBSCT v 57% BMT), nor were there any statistically significant differences in the occurrence of chronic GVHD (46% and 35%, respectively). There was no difference in death at 2 years between PBSCT and BMT (21% and 30 %, respectively, [P = .24]), whereas the incidence of relapse at 2 years was i4% and 25%, respectively, (P = .04). For overall and disease-free survival at 2 years, the percentages were 66% and 54% (PBSCT and BMT, P - .06) and 65% and 45% (PBSCT and BMT, P = .03). In the subgroups of patients with less advanced versus more advanced cancers, there was no difference between PBSCT and BMT in those with iess severe disease for 2-year survival. For those with more advanced disease, however, there was a 57% survival rate