Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
From www.bloodjournal.org by guest on June 15, 2017. For personal use only. CONCISE REPORT Late Expression of M and During By Marja The M/N cell sialoglycoprotein. drate blood groups is needed due for to amino of the acid for carried the A. activity. but We M and N blood HE M AND carried to study of the human protein, erythrocyte B, glycophorin blood group. cells, group antigens major the The irrespective A is expressed proerythroblasts,2 and the corresponding the carbohydrate rin A appear identical, M/N antigenicities oligosacchanides because are Recently, monoclonal for such at which bone normal was essential for abolished by control stainings, rabbit routine normal human examinations Alternatively, thoracic pieces surgery source of bone at of bone marrow of ribs which Helsinki marrow by further Finnish typing Specific En(a - Cross rabbit red cell cell through of M and specific membranes, which which 8161 4(8A2) phonin A,9 respectively, against were Culture Collection (Rockville, tane (Sigma, St Louis)-treated 1 (July). lack produce 1985: from described.3 The MGG. of the monoclonal rabbit anti- (FITC)-conju- mouse first IgG and normal antibodies. of antibody of erythroid cells, cells were irrelevant the centrifuged monoclonal binding bone cells were was counted. onto lgG antibody or antiserum of staphylococci bound to different marrow cells were was glass slides or normal used in control least 50 cells stained Ig instead rosettes. to the morphologically At and rabbit The recognizable of each maturation scored. labeled. oxidation with the cell-surface Sialic sodium acid residues metapeniodate glycoconjugates were were radiolabeled at 0 #{176}C followed by by reduction with NaB3H4.’2 Immunoprecipitations of the surface-labeled membrane lysates were made with monoclonal antibodies 8 I 59 or 8 161, followed by rabbit anti-mouse Ig using the protein A containing S From by the prepared N forms American as with A. The the Pathology healthy hy8159 of glycoType Md). Ascites fluid produced in PnisBALB/c mice by the 8159 clone- pp 233-236 the with were absorption antibodies the with were immuno- . Transplantation and No. by and 5 ROl the CA Pathology. Helsinki, © I 985 University Finnish theSigrid 26294-05 reprint from Society, Foundation, the University of to Dr Le:fC. Helsinki, Departments the of Academy Helsinki, National 25, 1985; accepted requests and of Helsinki. Cancer Juselius Bethesda. Md. Submitted March Address Laboratory Biochemistry, Supported Finland. glycophonin M and obtained from was by Double myeloid performed monoclonal the density cells Helsinki. A antiserum clones No was were smears stained smears minutes. isothiocyanate analysis stages radioactively a England) were treated in suspension with rabbit anti-glycophorin A antiserum or the monoclonal antibodies 8159 and 8161 Cells with antibodies bound to their surface antigens were allowed to form rosettes with protein A containing Staphylococcus aureus Cowan I strain as column.” consent Service, as cells Sweden) Ig-anti-Ig N antigens rendered suspended erythroid informed Transfusion were containing and and Vol 66, the an with used mononuclear Uppsala, cases, were Cheshire, cytocentnifuge monoclonal instead semiquantitative stage open of clone- of 1:5,120. the slides by incubation Fluorescein For immunoprecipitation, during cells the anti-glycophonin (6A7) Blood, marrow during used maturational transplantation. resected of Fc-receptor obtained Blood described3 ) removal obtained Hospital and some by passage The Red previously bnidoma cells were volunteers. In by the samples before been (Pharmacia, enriched monocytic donors bone (PBS), Ficoll-Isopaque centnifugation.’#{176} Blood were had The saline gradient and marrow University cells. in phosphate-buffered isolated bone the followed irrelevant Ig were number METHODS incubating or 8161 dilution 8161 gated sheep anti-mouse lgG and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated sheep anti-rabbit Ig antisera (Cappel Laboratories, Cochranville, Pa) were used as second antibodies. In For by sialylation.8 by the 20 #{176}C) for ten - a cells were prepared Products, staining, ( made at by the at a dilution Southern A antiserum. erythroid from (Shandon 8159 glycophonin of the Cells erythrocytes produced from the bone marrow immunofluorescent For with AND type ascites N erythrocytes smears a Shandon antibodies marrow. MATERIALS and homozygous staining antibodies which recognize epitopes have become available.9 We have to determine the stages of erythroid the M and N antigens are expressed in antibodies findings Inc. M 1:10,240, fixed in cold methanol M and N antigens maturation These For morphological analysis, with May-Grunwald-Giemsa (MGG). ery- marker are these & Stratton, homozygous to using precursors.34 the M and N antigens polymorphism within of sialic acids and can be regained removal used the was abun- recognizable proerythroblast. cytocentrifuge. positions.5 Although the compositions of moieties of the M and N types of glycopho- the by Grune Cytocentnifuge terminal 5, while the it already maturation. 1985 S agglutinated surface recognizable it is a useful erythroid M/N region of the glycophorin A polypeptide.5 AM contains serine and glycine at positions 1 glycophorin AN has leucine and glutamic acid at the amino Glycophorin on the the precursor. 1:5,120 sialoglycoof the on morphologically both normal and malignant erythroid The structural differences between are based on the amino acid sequence and minor dantly agglutinated primarily sialoglycoprotein N-active antiserum. be explained by our previous observation that the 0-glycosylation of glycophorin A gradually increases during expression are A glycophorin A molecules are present on the earliest morphologically can we report that the M/N weakly or not at all normoblast stage. A, anti-glycophorin that erythroid antibodies membrane.’ is Glycophorin of the earliest membrane throid blood N is portion their polyclonal shown specificity NH2-terminal A and Leif C. Andersson Using red carbohy- monoclonal groups by glycophorin human M/N the used during normal erythropoiesis. Here blood group activities are very expressed before the polychromatic T major on Glycophorin Differentiation Carl G. Gahmberg, 0-glyosidic in the have Erythroid Ekblom, by the replacements molecule. specific are glycophorin N Antigens April and Institutes of of by grant Health. 22. 1985. Andersson. Haartmaninkatu Department 3. SF of 00290 Finland. by Grune & Stratton, Inc. 0006-4971/85/6601-0035$03.00/0 233 From www.bloodjournal.org by guest on June 15, 2017. For personal use only. 234 EKBLOM, Cowan I strain.’3 Electrophoresis on 8% polyacrylamide slab gel was performed according to Laemmli.’4 The gels were fixed and treated for fluorography as described.’5 GAHMBERG, AND ANDERSSON aureus ‘ A .t RESULTS -....e A E% homozygous with the Surface-labeled immunoprecipitated and 8161 and were identified the molecules recognized by polyacrylamide slab homozygous no bands N red cells, anti-M (8 1 61 ), were obtained M and N erythrocytes monoclonal antibodies antibody (Fig the glycophorin from were precipitated with the anti-N and dimer N red cells to the dimeric B from phorin both (Fig and monomeric 1C), forms cells by analyzed (Fig 2). strongly throid (Fig 3). of glyco- basophilic more mature both the and A Cowan and the weak normoblasts. erythroid technique the rabbit anti-glycophorin anti-N antibodies. The ery- and anti-N proerythroblasts Polychromatic showed reacted normoblasts anti-M with cells was recognizable monoclonal reactivity marrow antiserum basophilic normoblasts strong reactivity and with the anti-M the same and stage In indirect A antiserum (Fig 4A). reacted nucleated immature the anti-N antibodies of the erythroid appeared The monoclonal at approximately differentiation. immunofluorescence, stained large nucleated anti-M rabbit anti-glycophorin erythroid precursors and anti-N antibodies with mature erythrocytes and relatively small red cell precursors (normoblasts), while the largest glycophorin A-containing cells did not stain with monoclonal antibodies A and the monoclonal epitopes recognized by BC (Fig 4B). 5o 40 R anti-GP-A ::. DE - x64). and the N antigens 1-rosetting anti-glycophorin A earliest morphologically contrast, showed anti-M S aureus Fig 2. The binding of monoclonal anti-N antibody 81 61 to bone marrow cells as assessed by the S aureus Cowan l-rosetting technique. Erythroid cells are marked by letters. A. proerythroblast; C, polychromatophilic normoblast; E. erythrocyte. MayGr#{252}nwald-Giemsa stain (original magnification x 1 00; current magnification M and the proerythroblasts, In antibodies and the Rabbit with the cells, for heterozygous ‘$ but M and N red cells. The binding of the anti-glycophorin A antiserum monoclonal anti-M and anti-N antibodies to bone erythroid 4 the antibody molecules not from MM red cells (Fig 1E). Similarly, the anti-M antibody reacted with glycophorin A from M cells (Fig I D). In addition, the anti-N antibody precipitated two bands corresponding “ by these antibodies gel electrophoresis. In 1B). Using A monomer homozygous were 8 1 59 - .- 10 0 z - ! 0 .il& 50 40 8159 0 GPA-D--- - GPA-D-- 30 >- 20 I 10 GPA-M GPB-M-- $ - O - GPA- -GPB-M M- $ - GPB-D -GPB-M Fig 1 . Polyacrylamide slab gel to show specificity of monoclonal anti-M and anti-N antibodies. (A) Surface glycoprotein pattern of RBCs labeled by the periodate/NaB3H4 technique; (B) pattern of immune precipitate obtained from 3H-labeled NN cells with anti-M antibodies; (C) pattern obtained from 3H-labeled NN cells with anti-N antibodies; (D) pattern obtained from 3H-labeled MM cells with anti-M antibodies; (E) pattern obtained from 3Hlabeled MM cells with anti-N antibodies. GPA-D. glycophorin A dimer; GPA-M. glycophorin A monomer; GPB-D. glycophorin B dimer; GPB-M. glycophorin B monomer. Note that GPA-M and GPB-D have the same electrophoretic migration rates and that GPB is N-active irrespective of the MN blood group. Aa Bb Cc Dd Ee Fig 3. The binding of rabbit anti-glycophorin A antiserum (R anti-GPA-A) and monoclonal anti-M (81 59) and anti-N (8161) antibodies to bone marrow erythroid cells. Vertical bars indicate the number of staphylococci per cell (mean ± SD). Aa. proerythroblast; Bb. basophilic normoblast; Cc. polychromatophilic normoblast; Dd. orthochromatic normoblast; Ee. erythrocyte. Capital letters indicate binding of the relevant antibodies and small letters indicate binding of control immunoglobulins from the same species as the antibodies. From www.bloodjournal.org by guest on June 15, 2017. For personal use only. LATE EXPRESSION OF M AND 235 N ANTIGENS that the anti-N antibody (8161) N types of glycophorin.9 We reactivity reacted Instead, AM. precipitated glycophorin In addition, any crossof this antibody with the 8161 antibody specifically anti-N antibody B from described the AN. the monoclonal glycophorin earlier N erythrocytes rin B, whose both from M and N types immunoblotting precipitated of erythrocytes studies.9 as Both contain some N antigen activity first 23 amino acids are identical glycophorin the M and both not observe in immunoprecipitation glycophorin with did M and in glycophowith those of AN.S Glycophorin A has been shown to be a specific and early erythroid marker present in the membrane of proerythroblasts.23 The epitopes and anti-N antibodies clearly cells later, by the monoclonal anti-M on the surface of erythroid recognized appear at the polychromatic normoblast demonstrated aureus Cowan Carbohydrates by the double immunofluorescence I-rosetting technique. are known to contribute determinants recognized by the is highly glycosylated, monoclonal The relatively determinants, phorin Fig 4. erythroid Double cells. immunofluorescence (A) Cells staining stained with of bone rabbit marrow anti-glycophorin A antiserum followed by TRITC-conjugated sheep anti-rabbit The same field stained with monoclonal anti-M antibody followed by FITC-conjugated sheep anti-mouse lgG. 1g. (B) 8159 glycophorin are rin molecules A The monoclonal to react specifically findings from the and N erythrocytes . VT, Sci USA of the Robinson observations. and reported consistent changes erythroid with early of than by the rabbit reflects those from antigens monoclonal phorin anti-glycophorin in the glycosylation The present maturation. our recent erythroid more the present results leading to expression Since changes adequate Sieff Andersson Glycophorin tiation in acute 5. Furthmayr immunochemical 1978 surface orientation erythrocyte C, Delia Segrest JP, Scott of the membrane. D, HLA-DR, differentiation. Proc 52:379, LC, NatI RE: major Acad finding precursors mature that glycophoare less 0- erythrocytes.” show that the carbohydrate of the M and N blood group relatively late during erythroid antibodies reacting with epitopes A to which carbohydrates contribute for phenotyping early erythroleukemias. maturation, on glycomay not be occur CG, marker Int J Cancer Structural analysis Greaves and M: glycophonin 1981 LC: Expression erythroid cells of Teerenhovi of early of the in human 23:717, comparison genetic variants. L, Vuopio erythroid P: differen- glycophonins Nature enzyme. M and N by influenza Proc NatI Acad Sadler JE, Paulson expression of human 254:2112, 1979 viruses Sci USA JC, MN 44:182, and 271:519, 6. M#{228}kel#{228} 0, Cantell K: Destruction of M and N blood group receptors of human red cells by some influenza viruses. Ann Med Exp Biol Fenn 36:366, 1958 7. Springer GF, Ansell NJ: Inactivation of human erythrocyte and receptor-destroying 1958 Hill RL: The role of sialic blood group antigens. J acid in the Biol Chem 9. Bigbee WL, Vanderlaan M, Fong 55, Jensen RH: Monoclonal antibodies for the M- and N-forms of human glycophorin A. Mol Immunol 20:1353, 1983 B#{246}yum A: Isolation of mononuclear from human blood. Scand J Clin Lab Invest 10. 1979 of agglutinogens 8. 1978 surface leukemia. H: on Gahmberg A as a cell PAW, 289:68, M, Andersson (glycophonin) Blood Edwards HLA-ABC Nature CG, Jokinen sialoglycoprotein bone marrow. RL, and of cell-surface erythroid Jackson 1972 J, 3. Gahmberg 4. TW, human 69:1445, Expression major Tillack characterization glycoprotein during with these technique identified REFERENCES Marchesi Chemical 2. 8159 has been reported of glycophorin A.9 Our of homozygous M are in agreement an immunoblotting Bigbee et al used 1 anti-M antibody with the M type immunoprecipitation of blood group M and N to the appearance of the glyco- A during results oligosac- oligosaccharides.’6 expression apparently glycosylated DISCUSSION late 1 5 0-glycosidic as compared A molecule A antiserum, of and NH2-terminal mature red cells N-glycosidic one containing at ASN-26 as the S to the antigenic M/N-specific antibodies 8159 and 8l6l. The external, portion of the glycophorin A molecule from charide stage and 1 1. Wigzell using glass (suppl 5) 12. Gahmberg of cell surface J Biol Chem 13. human I 978 H: or plastic Specific bead affinity columns. CG, Andersson sialoglycoproteins 252:5888, 1977 cells and granulocytes 21:77, 1968 (suppl 97) fractionation Scand of lymphocytes J Immunol 5:23, 1976 LC: Selective radioactive labeling by peniodate-tnitiated borohydride. Gahmberg CG, Andersson LC: Leukocyte surface a,-acid glycoprotein (orosomucoid). J Exp Med origin 148:507, of From www.bloodjournal.org by guest on June 15, 2017. For personal use only. 236 EKBLOM, 14. Laemmli assembly 15. tnitium of the Bonner labelled Eur J Biochem 16. Tomita UK: head Cleavage WM, Laskey proteins 46:83, of structural of bacteriophage and T4. RA: A film nucleic acid proteins Nature during 227:680, detection the 1 970 method in polyacrylamide NatI for gels. 1974 M, Marchesi chanide Amino acid sequence and oligosac- Sci 17. Gahmberg human the VT: attachment Acad USA erythroid major sites USA human 1984 erythrocyte AND ANDERSSON glycophonin. Proc 1975 CG, Ekblom cells M, Andersson is associated sialoglycoprotein, 81 :6752, of 72:2964, GAHMBERG. with glycophorin LC: Differentiation increased 0-glycosylation A. Proc NatI Acad of of Sci From www.bloodjournal.org by guest on June 15, 2017. For personal use only. 1985 66: 233-236 Late expression of M and N antigens on glycophorin A during erythroid differentiation M Ekblom, CG Gahmberg and LC Andersson Updated information and services can be found at: http://www.bloodjournal.org/content/66/1/233.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.