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From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Canine CD34: Cloning of the cDNA and Evaluation of an Antiserum to Recombinant Protein By Peter A. McSweeney, Katherine A. Rouleau, Rainer Storb, Laura Bolles, Philip M. Wallace, Mary Beaucharnp, Ljiljana Krizanac-Bengez, Peter Moore, George Sale, Brenda Sandrnaier, Thierry de Revel, Frederick R. Appelbaurn, and Richard A. Nash Increasingly, enriched populations ofhematopoietic progenitors are used in experimental and clinical transplantation studies. The separation of progenitors is based on the expression of CD34, a marker preferentially expressed on progenitor cells. The dog modelhas been important forpreclinical transplant studies, because it has proven predictive for outcomes in human hematopoietic stem cell transplantation. To identify and isolate canine hematopoietic progenitors, we have cloned a cDNA encoding a CD34 homologue from a canine myelomonocytic leukemia cell line, ML2. The CD34 homologue cDNA predicts an amino acid sequence that is highly conserved with human and murine CD34 in the cytoplasmic domain, transmembrane domain, and C-termina1 end of the extracellular domain, but shows considerable divergence from these sequences at theamino-terminal end ofthe protein. In Western blotting studies, canine CD34 homologue (caCD34) appears t o be a heavily and variably glycosylated protein with a molecular weight of approxi- mately 100 kD and shows some tissue-specific differences in protein mass. To evaluate the expression of caCD34 protein, the extracellular domain of caCD34 was expressed as an lg fusion protein andused as an immunogent o generate a rabbit polyclonal antiserum. The antiserum reactedagainst the fusion protein, against vascular endothelium, and with three leukemic cell lines. Approximately 1% of canine bone marrow cells stained brightly withantibodies t o caCD34 and 25- t o 50-fold enriched for colony-formthis population was ing units-granulocyte-macrophage as compared t o unfractionated marrowmononuclear cells. Thesefindings suggest that thecanine CD34 homologue is expressed on bone marrow progenitor cells and, thus, that this molecule should be a valuable marker for identifying and isolating canine hematopoietic progenitors for experimental hematopoiesis and stem cell transplantation. 0 1996 by The American Society of Hematology. C CD34 may be a ligand for leukocyte L-selectin with a possible in vivo role in vascular adhesion.'," The function of CD34 is not fully understood and human and murine CD34 have no close homology to other known proteins. The restricted expression of CD34 has allowed for antibodies to human CD34 and to murine CD34 to be used to identify and enrich hematopoietic progenitors as determined by in vitro3.1?.llandin vivo studies.""'In humans, monoclonal antibodies (MoAbs) to CD34 recognize about 1.5% of marrow mononuclear cells' and CD34-enriched progenitor cells, derived from marrow or peripheral blood stem cells ( P B X ) , have the capability to fully restore hematopoiesis after treatment with myeloablative chemoradiotherapy.""' CD34 selection technology is an active area of clinical researchas a method for purging autografts of tumor or T-cell-depleting stem cell allografts to prevent graft-versushost disease (GVHD). The dog has a proven role in experimental marrow transplantation and results of canine transplantation studies have predicted findings that have led to important advances in allogeneic transplantation.'' Because no marker of early canine hematopoietic progenitors has previously been reported, we undertook to determine whether the canine homologue for CD34 (caCD34) would be such a marker. We describe here the cloning of a cDNA for caCD34 and the characterization of cells expressing this protein. D34 IS A TYPE 1 transmembrane protein expressed primarily on primitive hematopoietic progenitor and vascular endothelium from many tissues."' Cell surface expression of CD34 is developmentally regulated in hematopoiesis' and isinverselyrelatedto the stage of differentiation, such that CD34 expression islostbeyond the committed progenitor stage. This pattern of expression suggests an important role of CD34 in early hematopoiesis. The genes for human and murine CD34 have been cloned'-"' and have significant homology of the genomic structure. cDNA sequence, amino acid sequence, and predicted protein structure. Predicted structural features of these proteins suggest properties of an adhesion molecule of the sialomucin class of molecules" and murine studies showed that vascular From the Clinical Research Division, Fred Hutchinson Cancer Reseurch Center, Seattle, WA; the University of Washington, Seattle. WA; Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA; and the University of California, Davis, CA. Submitted Februap 26, 1996; accepted May 8, 1996. Supported in part by Grant No. DK42716from the National Institute of Diabetes and Digestive and Kidney Diseases and Grants No. CA47748 and CA31787from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. Bethesda, MD. Support was also received from a prize awarded by the Josef Steiner Krebsstifiung, Bern, Switzerland. P.A.M. is a FeIlow of the Leukemia Society of America. L. K.-B. is supported by the Department of Experimental Biology and Medicine, Rudjer Boskovic Institute, Zagreb, Croatia. Address reprint requests to Peter A. McSweeney, MD, Fred Hutchinson Cancer Research Center, I124 Columbia St, M318 Seattle, WA 98104. The publicationcosts of this article were defrayed in part by page charge payment. This urticle must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. 0006-497//96/~806-0047$3.00/0 1992 MATERIALS AND METHODS Cell lines and cell culture. The canine leukemia cell lines MLI , l y ML2, ML3 (all myelomonocytic), 1390 (CD8' leukemia) (ML2, ML3, and 1390 were derived from spontaneous canine leukemias and provided by P. Moore, University of California, Davis, CA), and CLGL 90 (large granular cell leukemia; M. Wellman, manuscript in preparation) were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 25 m o V L HEPES, 0.1 mmol/L minimum essential medium (MEM) nonessential amino acids, I mmol/L sodium pyruvate, and 0.05 m o l n 2-mercaptoethanol. Jugular vein endothelial cells from normal dogs were purchased Blood, Vol 88, No 6 (September 15), 1996: pp 1992-2003 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. CLONING OF A DNA FOR CANINE CD34 1993 A Coding sequence ....................................... cDNA 0 - C 0 c A B D 44-3 28-1 54-1 Exon X B Fig 1. (A) Map of the cDNA clones for caCD34 in relationship to t h e proposed cDNA sequence. Gray segments at each end of the cDNA line represent sequence obtained from genomic clones. The alternatively spliced exon is shown as exon X from clone 5-41. The cDNA fragments isolated by PCR for subcloning and sequencing were flanked by the primer pairs (1)34A (A) and 348 (B) and (2) 34C (C) and 34D (D). (B) Genomic map of caCD34 showing location of phage clones and predicted exon-intron locations with exons numbered underneath. The genomic segment 3‘ of the phage clones was determined by restriction analysis of canine genomic DNA. Constructs made for sequencing t h e 5‘ and 3’ ends of t h e cDNA are also shown. Known restriction sites are shown as vertical lines with abbreviations as follows: S,Sa/ I; E, EcoRI; B, BamHI; SM, Sma I; SC, Sac 11. SM BC E B B BEBE B BE I IIII II I U 111 I 2 3 4 1 EB B I E l - 1 kb III= 567 a CD%-27 CD34-14 CD34-5 CD34-2 B 7.0kb B 5.0 kb S S S-SC S- -E E 3.8kb 1.4kb SM 1.3 kb from Endotech (Indianapolis, IN) and cultured according to the manufacturer’s instructions. Northern Alntting. Total cellular RNA was prepared from cell suspensions by guanidinum isothiocyanate lysis’“ using RNAzol A (Texas Biotech, Houston. TX). Fifteen or 30 pg ofRNA was run on a I % glyoxal denaturing agarose gel and transferred to nylon membranes. Membranes were probedwith a human CD34 cDNA probe (kindly provided byDrD. Tenen. Harvard Medical School, Cambridge. MA) radiolabeled with [a-”P]dCTP (NEN Dupont, Boston, MA) using the random hexanucleotide priming method.” Reverse trc~nscription-po!\rnercrse clwin reuctinr~ ( R T - f CR). PCR primers 34A and 348 were designed from sequences of highest homology when comparing humanand murine CD34 cDNAs. Primers were (see Fig I A ) (a) 34A (sense) S’-CCGAATTCGCTCCTTGCCCAGTCTGAGG; (b) 34B (antisense) 5”CCGAATTCCACGTGTTGTCTTGCTGAATGG: (C) 34c (sense) ~ ” T A G A A G (I7T(JTCGAGAAGGATGCGGCGGGCA; and (d) 34D (antisense) S’-CAGAATTCACTGTGGTAGGAGTAATCAC. EcoRI (A, B, and D) and Hind111 (C) restriction sites (underlined) were included. Reverse transcription andPCR were performed as previously described.” Samples were electrophoresed through 2% agarose gels and analyzed by ethidium bromide staining andor by probing of Southern blots with caCD34-specific [y-”PIdATP end-labeled oligonucleotides. PCR products were cloned into the EcoRI restriction site of pBluescript (Stratagene, La Jolla, CA) or directly into pT7Blue T-Vector (Novapen, Madison, WI)” and sequenced as described below. To extend the S’ end of thecDNA, rapid amplification of cDNA ends’‘ (S’ RACE) was performed using two commercial kits (GIBCO BRL [Gaithersburg, MD]and Clontech [Palo Alto, CA]) as described in the instructions from the manufacturers. Screening of canine cDNA und genomic phage lihruries. A random-primed cDNA library was made in Lambda ZAP (Stratagene) frompoly-ARNApurifiedfromtheML2 cell line. Inserts were cloned nondirectionally into EcnRl restriction sites. Nylon filters (Amersham. Arlington Heights. IL) lifted from plates containing 1.2 X IO” plaques were probed in buffer containing SO% formamide at 42°C with caCD34-specific probes radiolabeled by random hexamer priming. Filters were washed at a final stringency of either O.SX or 0.1x SSPE at42°Cand exposed toKodakXARfilm (Eastman Kodak.Rochester. NY) at-70°C for 1 to 3 days. Phage eluates from positive clones were evaluated for insert size byPCR using 34A and 34B or gene-specific primers (GSP), combined with vectorspecific RNA-polymerase site primers, T3 or T7. Clones selected for further characterization were converted to plasmids [pBluescript SK(-)] byanin vivo excision.’5 A canine genomic phage library made in Lambda Fix I1 (Stratagene) was screened with a cDNA clone and then with genomic subclones. Clones were characterized by restriction enzyme mapping. Southern blotting with GSP oligonucleotides. andPCR using primer pairs predicted to be located in different exons of caCD34. Clones were sequenced on both strands usingthe dideoxynucleotide method’“ or by PCR-based cycle sequencing withtheTaqDyedeoxy terminator cycle sequencing kit (Applied Biosystems. Inc. Foster City. CA) using an Applied Biosystems 373A DNA sequencer. Sequence analysis was performed with Genepro 4.2 software (Riverside Scientific Enterprises, Bainbridge Island. WA) and software from Genetics Computer Group (GCG) Inc (Madison, WI). froduction of recomhincmr cuCD34. An expression construct for the extracellular domain of caCD34 was made byPCR using primers 34F (93 bp; sense, SAGGAAGCTTCTCGAGATGCTGGTCCGCAGGGGCGCGCGCGCAGGGCCCAGGATGCCGCGGGGCTGGACCGCGCTTGCCTGCTCAGTCTGCTG: amino acid sequence, MLVRRGARAGPRMPRGWTALCLLSLL) with S’ Hind111and Xhn I restriction sites (underlined) and34G (32 bp, antisense). 5’-CTCCAGATCTGGCTTGCGGGAATAGCTCTGGT with a BgI 11 restriction site (underlined). The PCR productwas cloned into an expression plasmid containing a murine Igheavy chain sequence. A caCD34-murine Ig fusion protein (CD34-Ig) and a control fusion protein (murine CTLA4Ig containing the same murineIg sequence) were produced by transient expression in COS cells and purified as previously described.” Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) wasperformed From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 1994 withprecast 4% to 20% gels (Novex, San Diego, CA) using the manufacturer’s instructions. Automated N-terminal sequencing was performed at the Universityof Victoria Microanalytical Centre (British Columbia,Canada)using an AppliedBiosystemsmodel473 pulsed liquid sequencer. Immunization of rabbits and production of afJinity-pur$ed untiCD34 polyclonal antiserum (RPacaCD34). A female New Zealand white rabbit was used to generate a polyclonal antiserum to caCD34 as follows. Primary immunization was with combination of 2 mg CD34-Ig in 250 pL phosphate-buffered saline (PBS), 2.50 y L seppic montanide ISA SO, and RIB1 adjuvant injected subcutaneously in SO+L aliquots at 10 sites. The animal was boosted at 1 month with 1 mg CD34-Ig, at 6 months with ML2 cells, at 8 months with ML2 cells, and at I O months with a combinationof ML2 cells and canine endothelial cells from primary culture. Rabbit IgG from serum was purified on immobilized recombinant protein A (IA-300; Repligen, Cambridge, MA). The subfraction of IgGspecificforCD34was then isolated in two steps. First, IgG reactive with the IgG tail of theimmunogenwasremoved by adsorptiontoacolumn of the immobilized control fusion protein (CTLA4-Ig). Second, IgG reactive to CD34-lg was affinity-isolated on a column of immobilized CD34-lg. Columns were preparedby immobilization of protein onto CNBr-activated Sepharose 4B using the manufacturer’s instructions (Pharmacia Biotech, Uppsala, Sweden). Anti-CD34enzyme-linkedimmunosorbentassay(ELISA). The anti-CD34 ELISA was performed as described previously”with the following modifications. Immunlon 2 (Dynatech) flat-bottom plates were coated with 3 pg/mL CD34-Ig diluted in 0.05 mollL bicarbon9.6. Boundantibodywasdetected with a atebindingbuffer,pH 1:8,000 dilution of horseradish peroxidase-conjugated antirabbitIgG antibody (Southern Biotechnology, Birmingham, AL). Westernblorting. CellswerewashedinPBSandlysed with a solutionof I % NP40, 1.50 mmol/LNaClin 50 mmollLTris, pH 8.0. Nuclei were removed by I O minutes of centrifugation at 10,OOO g and lysates were stored at -70°C. Lysates were electrophoresed in a reduced Trislglycine SDS 8% polyacrylamide gel (Novex). Protein was transferred to a precut polyvinylidene difluoride (PVDF) membrane (Novex) using a wet Western transfer apparatus (Novex) in Tris-glycine transfer buffer. Membranes were blocked in 5 mg/ mL nonfat dry milk in PBS and then incubated with either RPacaCD34 or control rabbit serum followed by incubation for I hour withgoatantirabbitalkalinephosphatase(BoehringerMannheim Biochemicals, Indianapolis, IN) diluted in blocking solution. Membranes were washed four times with TTBS (2.5 mmol/L Tris, IS0 mmol/L NaCI, 0.5% Tween-20, pH7.5) and proteins were visualized with Western blue AP substrate (Promega). Flow cytometry. The followingantibodies were used: RPacaCD34, SS” (murine IgGl MoAb, anti-CD44. as apositive control), 31A’” (a murine IgGI, nonreactive with canine hematopoietic cells), and normal rabbit serum. RPacaCD34 wasused at 10 pg/mL, rabbit serum at a dilution of 1: 100, and SS and 31A at S yg/mL. A fluorescein isothiocyanate(F1TC)-conjugated polyclonal goat-antirabbit antibody (Caltag, San Francisco, CA) or a phycoerythrin (PE)-conjugated polyclonal goat-antirabbit antibody (Southern Biotechnology) and an FITC-conjugated goat-antimouse polyclonal antibody (Caltag)were used assecond-stageantibodies.Ficoll-Hypaque-separated bone marrow mononuclear cells (BMMC) and peripheral blood mononuclear cells adjusted to S to 10 X 10‘ cells/mL were stained for 20 minutes at 4°C at each stage and washed with PBS/2% horse serum.Forsomeexperiments,100-pLaliquots of unfractionated marrow or blood were stained after the addition of SO y L of PBS/ 2% horse serum and washed after each stage with PBS/2% horse serum, andthen erythrocytes were lysed with hemolytic buffer. Cells were fixed in 1% paraformaldehyde before analysis. Cell lines and cultured endothelial cells were incubated with RpacaCD34 ( I O ygl McSWEENEY ET AL mL), CD34-lg (100 pg/mL), or a combination of the two. Cells were thenwashedwithPBS,incubatedwithFITC-conjugatedsecondstage antibodies, and washed with PBS. Flow cytometry was perFACStar formed on a FACScan (Becton Dickinson, San Jose,orCA) (BectonDickinson),andthe list modedatawereanalyzed using Repromansoftware(TrueFactsSoftware Inc, Seattle, WA) and Cellquest Software (Becton Dickinson). Colony-forming unit-grclnulocyte-macrophage (CFU-GM)ussays. BMMC (2 X 107/mL) were stained with RpacaCD34 as described above, resuspended in PBSl28 horse serum, and sorted on a FACStar (Becton Dickinson). Lineage depletion of canine BMMC was performed with the following MoAbs: JD3 (anti-CD8):’ 1 E4 (antiCD4),3’ Tuk4 (antimonocyte; DAKO, Glostrup, Denmark), and DM5 (antigranulocyte),’2 using the magnetic activated cells sorter (MACS; Miltenyi Biotec. Sunnyvale, CA) separation system as per the manufacturer’sinstructions.Sortedandimmunomagneticallyseparated cell fractions were washed twice and 500, lo’, S X IO’, or IO4 cells per plate were assayed for granulocyte-macrophage progenitor cells ( C m - G M ) , as previously described.’’ Immunoperoxidase staining of tissuesections. Normaldog tissues were snap frozen in liquid nitrogen and 6-pm sections were cut onto glass slides using a Tissue Tek cryostat (Miles Scientific, Naperville, IL). Air-dried slides were fixed in acetone and in formyl calcium, and staining was performed using a TechMate I000 Immunostainer (BioTek Solutions, lnc, Santa Barbara, CA). Sections were treated with 5% goat serum/2% bovine serum albumin (BSA) fraction (Calbiochem, La Jolla, CA), Tween-20, and 0.4% sodium azide. Staining with RPacaCD34 (2.5 pg/mL) was detected by sequential applicationsofbiotinylatedgoat-antirabbit(Vector,Burlingame. CA) and horseradish peroxidase-streptavidin (Zymed, San Francisco. CA) followed by diaminobenzidine (Polysciences, Warrington, PA) at 0.5 mg/mL, 0. I % NiCl?, and 0.01 % H?OZ. Slides were counterstained with 0. I % acridine orangelo. 1 % safronin 0 and dehydrated rapidly through graded alcohols. As negative controls, staining was performed on canine tissues without primary antibody on andhuman lung to assess nonspecific tissue reactivity of RPacaCD34. A rabbit polyclonal antihuman factor VI11 (Dako, Carpinteria. CA) was used as a positive control to identify endothelial cells. RESULTS Isolation of the cDNA for caCD34. In Northern blotting studies using ahuman CD34 probe, the ML2 cell yielded line a transcript of approximately 2.8 kb, slightly larger than that from the KG1 myeloidleukemia cell line (human), which is approximately 2.6 kb.R This transcript was not detected from MLI or BMMC. In RT-PCR studiesusing primers 34A and 34B, a strong amplification signal of approximately 450 bp was detected from both KG1 and ML2, whereas weak signals were detected from MLI and canine bone marrow, consistentwithNorthernblottingresults. Sequence of the PCR fragment isolated from ML2 was highly homologous with 3’ coding sequenceof human and murine CD34 cDNAs and this fragment wasused as a canine-specific CD34 probe to screen the ML2 cDNA library. Initial screening yielded 60 positive clones and, based on PCR analyses of the insert sizes three clones, 54-1, 44-3, and 28-1 (Fig lA), were further characterized. Clone 54-1 extended 500 bp further 5’ than any other clone and contained a 3‘ 135-bp insert not found in the other two clonesthat suggested the presence of an alternatively spliced transcript. Clone 54-1, when used to probe the Northern blot previously described, hybridized to transcripts from ML-2 and KG-l of identical size to those From www.bloodjournal.org by guest on June 16, 2017. For personal use only. CLONING OF A DNA FOR CANINECD34 that hybridized to the human CD34 probe (data not shown). Attempts to extend the 5‘ sequence by rescreening the cDNA library with a 500-bp 5’ cDNA probe from clone 54-1 and with 5’ RACE were unsuccessful, suggesting the presence of extensive 5‘ secondary structure in the RNA molecule. Additional cDNA sequence for the signal peptide and 5‘ untranslated regionwas obtained after screening a canine genomic library withclone 54-1. Two unique clones, CD3427and CD34-14, together extended from 5’ of predicted exon 2 to 3‘ of predicted exon 8 (Fig lB), but neither hybridized to the exon 1 oligonucleotide 34E (antisense), 5’-CAGCAGACTGAGCAGGCAGAG, designed from clone 54- 1. A 3.8-kb EcoRI subclone from CD34-14 wasused to sequence the 3‘ end of the cDNA, including the polyA signal. After rescreening the library with a 5’ 1.1-kb BamHI fragment from CD34-27, two clones, CD34-2 and CD34-5, hybridized to the oligonucleotide 34E (Fig 1B). Becauseof the very high GC content in the presumed first exon, deletion constructs of a 5.0-kb Sal YBamHI subclone from CD34-5 were made to facilitate sequencing (Fig 1B). Also, a 131bp PCR cDNA fragment amplified from ML2 cDNA with primers 34C and 34D (Fig 1A) was sequenced to determine the signal peptide nucleotide sequence. Based on restriction mapping and PCR studies of phage clones, the likely genomic structure of caCD34 was determined (Fig 1B). The nucleotide sequence of the caCD34 cDNA, derived from both cDNA and genomic clones, is shown in Fig 2. The canine cDNA is 135 bp longer than the human CD34 cDNA, consistent with thetranscript sizes found by Northern blotting. The transcription start site has not been formally mapped, butis predicted from computer alignments of canine sequence with human CD34 cDNA and promoter sequences. Additional sequence 5’ of theproposedcDNA sequence (datanot shown) showedhomology to thehumanCD34 promoter sequence,34and also showed a TATA box motif 34 bp upstream of the proposed start of transcription. Exon 1 (nucleotides 1 through 338) wasextremelyGCrich (82.5%) and may be able to form stem-loop-stem structures that have been implicated intranslational regulation. A variant polyadenylation signal, AAUUAA,35started at nucleotide 2730 and is identical to the poly A signal seen in the human gene. Several AU motifs (AUUUA, AUUUUUUUA) that may have a role in mRNA stability were found in the 3’ untranslated region.36 Analysis of the predicted amino acid sequence. The main open reading frame of the cDNA is 1167 bp and began with the start codon AUG at nucleotide 260 and ended with the stop codonUGA (Fig 2A). This encodes a protein of 389 amino acids with a predicted molecular weight of 41 kD that has thefeatures of a type one transmembrane protein. The matureproteinwaspredictedusing SIGCLEAVE (GCG) to begin with either the asparagine at residue 32 or the glutamic acid at residue 34. An extracellular domain of 260 to 262 amino acids is characterized by an amino-terminal region of approximately 150 amino acids with a high serine and threonine content and with the potential for extensive 0-linked glycosylation. There are 7 potential N-linked glycosylation sites in the extracellular domain, of which 5 are found within this N-terminal region. The C-terminal end of 1995 the extracellular domain contains 6 cysteine residues between aminoacids 182 and 247, whichsuggests that disulfide bonding maybe important for structure of the protein. A 23 amino acid transmembrane domain extending between residues 294 and 3 16 is predicted based on the presence ofa hydrophobic domain and homologyto the human and murine sequences. The cytoplasmic domain is predicted to be 73 amino acids and has two protein kinaseC (PKC) phosphorylation consensus sequences3’ and a potential tyrosine phosphorylation site3’ at amino acid 334 (Fig 2A). Translation of the sequence from clone 54-1 (Fig2B) encoded an in-frame early stop codon for themainopen reading frame that results in a short cytoplasmic domain of 16amino acids, rather than 73 aminoacidspredicted by sequence from clones 44-3 and 28-1. Competitive RT-PCR studies were performed to evaluate the presence andrelative quantity of the two caCD34 transcripts using primers that flanked sequence encoding both transcripts (Fig 1A). Analysis of the ML2 line, several canine tissues, cultured endothelial cells, and bone marrow (data not shown) showed thatin each cell type the alternatively spliced transcript represented no more than 10% of the mRNA for caCD34. A search of the Genbank and Protein Identification Resource (PIR) databases did not identify any proteins with significant homology to caCD34 apart from the two CD34 sequences already reported. A comparison of the amino acid sequence from dog with those of the human and mouse (Fig 3) showedan overall aminoacidhomologybetweendog and human of 69% and between dog and mouse of 62%. Homologies of the cytoplasmic and transmembrane domains between the three species were high (>86%), whereas homologies in the extracellular domain weremuchlower: dog:human, 60%; dog:mouse, 55%; and human:mouse,56%. The overall structure of mature CD34 proteins appears very similar among these species, with the N-terminal portion of the extracellular domain being rich in serine and threonine residues. However, sequence homology is lower in this Nterminal region than further towards the C-terminal end of the molecule. The C-terminal 100 amino acids of the extracellular domain are cysteine rich, with the number andlocation of the cysteine residues being highly conserved; this suggests that the structure of these proteins maybevery similar. The transmembrane and cytoplasmic domains are highly conserved, including cytoplasmic PKC and tyrosine phosphorylation motifs, which suggest conservation ofintracellular signalling pathways. The three species all produce an alternatively spliced variant with an additional exon which, although different in size and nucelotide sequence, in each case encodes an identical 4 amino acids before a stop codon that truncates the intracellular domain. Expression and analysis of recombinant caCD34. To prepare antisera to examine the cellular expression of caCD34 protein, a fusion protein (CD34-Ig) was produced. Using primers 34F and 34G andclone 54- 1 as PCR template, a 906-bp fragment was amplified encoding a 99-bp signal peptide sequence and the 780-bp extracellular domain of caCD34 (Fig 4A). This fragment was digested with HindIII and Bgl I1 and subcloned into HindIII and BamHI sites of a 7rLN vectorthat contained cDNA sequence encoding the From www.bloodjournal.org by guest on June 16, 2017. For personal use only. McSWEENEY ET AL 1996 A.cccccctcggcctccagggcggcggcaaccccggcccccgctcccgtcccccgcctgcggggctgagccgagcgctcgcg 80 gtggcggcggccaagcggaggggccggccttgccaggaacgcggagggaggggtggggagagacagccagctcgcccacc 1 6 0 ccgctccgggcggagggcggagggcggcgggcggcgggcggcggcgcgtcccggggccgagcgcgtctgtccggagccga 2 4 0 gcggagcggcgcggGAAGGATGCTGGCGGGCAGGGGCGCGCGCGCGGGCGGCGGGCTGCCGCGGGGCTGGACCGCGCTcT 320 M L A G R G A R A G G G L P R G W T A L C 21 GCCTGCTCAGTCTGCTGCCCTTTGGGTTCACAAACACAGAAACCGTGATTACTCCTACCACAGTGCCAACCTCCACAG~ 4 0 0 A E T V I T P T T V P T S T E 47 ATAATGTCAGCTGTTTCTGAGAATACATCC~CGGGAAGCCATCACACTAACTCCTTCTGGAACTACCACCCTGTACTC 4 8 0 I M S A V S E N T S K R E A I T L T P S G T T T L Y S 74 TGTCTCTCAAGACAGCAGTGGGACCACAGCAACCATCTCAGAGACTACAGTCCATGTCACATCTACCTCTGAGATCACCC 5 6 0 V S Q D S S G T T A T I S E T T V H V T S T S E I T L 101 TAACGCCTGGGACCATGAACTCTTCTGTTCAGTCGCAGACCTCTTTAGCTATCACGGTATCTTTTACCCC~CCAACTTT6 4 0 T P G T M N S S V Q S Q T S L A I T V S F T P T N F 127 TCAACTTCAAGTGTGACCTTGGAGCCCAGCCTGCTACCTGG~TGGTTCGGATCCCCCCTACAACAGCACCAGCCTTGT 72 0 S T S S V T L E P S L L P G N G S D P P Y N S T S L V 154 GACATCCCCCACGGAATATTATACATCACTTTCTCCTACCCCAAGTAGAAATGACACCCCAAGTACCATCAAGGGAGAAA 8 0 0 T S P T E Y Y T S L S P T P S R N D T P S T I K G E I 181 TCAAATGTTCCGGAGTCAAAGAAGTGAAATTGAACCAAGGTATCTGCCTAGAGCTAAATGAGACCTCCAGCTGTGAGGAC 8 8 0 K C S G V K E V K L N Q G I C L E L N E T S S C E D 207 TTTAAGAAAGATAACGAAGACTGACCCAAGTCCTGTGTGAGAAGGAGCCAGCTGAGGCTGGGGCCGGGGTGTGCTC F K K D N E E K L T Q V L C E K E P A E A G A G V C S 960 234 CCTGCTTCTGGCCCAGTCTGAGGTGAGGCCTCACTGCCTGCTGCTGGTCTTGGCCAAC~CAGAACTTTTCAGT~C 1040 L L L A Q S E V R P H C L L L V L A N K T E L F S K L 261 T C C A A C T T C T G A G A A A G C A C C A G T C T G A C C T G A A A A A G C T C C A C 1120 Q L L R K H Q S D L K K L G I R D F T E Q D V G S H 287 CAGAGCTATTCCCGCAAGACCCTGATTGCACTGGTCACCTCAGGGATCCTGCTGGCTGTCTTGGGCACCACTGGTTACTT 1 2 0 0 Q S Y S R K T L I A L V T S G I L L A V L G T T G Y F 3 14 C C T G A T G A A C C G C C G C A G T T G G A G C C C T A C A G G A G A A A G G C G G T G G A G G C C 1280 L M N R R S W S P T G E R L G E D P Y Y T E N G G G Q 341 AGGGCTATAGCTCAGGCCCTGGGGTCTCCCCTGAGGCTCAGGG~GGCCAGTGTGAACCGTGGGCCTCAGGAGAACGGG1 3 6 0 367 G Y S S G P G V S P E A Q G K A S V N R G P Q E N G ACCGGCCAGGCCACGTCCAGAAACGGCCATTCAGCAAGACAACACATGGTGGCTGATACAGAATTGTGACTCTGGGGGGG1 4 4 0 389 T G Q A T S R N G H S A R Q H M V A D T E L GAGTAAGGCTGGGCAGGGTCTGGGGAAGGGGGCCCCTCCCAGCACCTGACCACATGCTGCCTCTGTGCTGGAGCTGCCACC 1 5 2 0 ACTTACATTCTAGCCTTTCCTGCTGCACACACCCTCCGAGGCCATTCCTGGGGCCCTGCACTGCACCAGGCCGAGGGGTT1 6 0 0 1680 CTCTCCATCCTGGGGCCCGGGAGGTAACCCCCTACCTTTTATACATTCATCTCACTAAGCCTAGAGTCTGGTCTCCTTTGA GAAAAGACATGAGGGAGACGTGCCAAAGTATAGAGAAGCTACCAGAGTTGGGGGGGTGGGGGGTGATGATCTCACATCAT 1 7 6 0 1840 TCACGTGTGGGCTTCTTCCCTCTTCCTCCTCTCTGCCTTATT~G~CTTCCAGGGGGAAGCATGGCCTTTTCTGGGC 1920 TACAATGTCCTCCTGGGAGGCTTTGTCTTTTCCTGTGTCTTCCTCATGTCTGTCTCCTCTACTTTAGGGAAACC~GCA CCTGCTCCTTTGTAATGCTATAGCCAGCAAGACTTGTTGTCTTAAACCGTCTCCCTTGTGCTCACACCAGCTCACTGTGG2 0 0 0 ATTCAGGCAACCGGCTTCCCTCATGCTCTCCGGGCTCCCTGAGCTCCACACCTTCTCCCTGCACCTCTGTGTACAGAC 2 0 8 0 CTGCACTGTTCTCTGGCTGAGCCTGGAACGAGACTCCAAGTTTTG~CAATGTCTTGTGTCTATGTTTGGGAGACAGCAT2 1 6 0 A G G G A T G C G T G G A C A C A T G C G T T C C T A T C T T T G G G G A C A A T C C T T G T C T C T C T G G G G 2240 CTCACAGAGTCTCATCTTGGGCCCCCGTTTCTCCCTGTGAGTCTCAGTGAACGGGACCAAGGGACCAGATCTTGGAGCCA 2 3 2 0 AGCCTCTTGACCCATGCACCTCTGAAGAAGCCCCTCGCTCGAAGGCTAGGTCCTGGCCTTGCCCTCTGATCCTGATGGCT 2 4 0 0 TCCTCCTTCCTCCCTCTGACTCCTGGGTGAGCTGTGGACTCAGACTCCCAGCAGACTCCTTTctgtctcagcctccccga 2 4 8 0 ccccaaccccctcactgttctgtac~cccatatagtcagggcccccgacatctccagaggaccttcatcacaagccatct 2 5 6 0 cctctgtaggtggcccaggttctcatttatttttttaggtattttt~ttttccagaggggtgagcagagatcttggtttc 2 6 4 0 aatgacggttggaaatagaactttccagagataggaagactgggtggattttatttctgaatacaaaaatggtgtgtgta 2 7 2 0 aatactgtaatta~agtgataccgagacacatctgttctgtgtcgctgccccagccaggtgtgtctgaatgccacggcgg 2 8 0 0 2880 tgtccctggtgtcccggtcagacccggccagacttcccaatgatgtggtagagaggggtgaccctggaaagaggtgggcc 2956 catctcgggggatacaggcaaaagccagggtgctgccccttggccaagtgtccctatgggtggggggggttggagg B.tggagccctacaggagaaaggctggAGCTGGAACCC~ATCGCTCTTCAGGAAG~GGAGTCT~CACATGCAGCTACAA5 5 W S P T G E R L E L E P CCCCCACTCTCTCCCCCACCCCCCACTGCCTCAATCCCCTGCTTACCAGATAATGCTCCTTTATTTATACACTGTCTAGG 1 3 5 Fig 2. Nucleotide sequence of cenine CD34 with predicted amino acid sequence shown underneath [GenBank accession no. U49457). The sequence shown in lowercase at each end of thecDNA was determined from genomic phage clones. The polyadenylation signal is underlined starting at nucleotide2729. Features of the aminoacid sequence are highlighted as follows. (A)33 amino acid leader peptide (underlined).the transmembrane domain litalics/underlined), highly conserved cysteine residues (bold), and potential N-linked glycosylation sites INXTIS; underlined). Potential PKC (double underlined) and tyrosine phosphorylation sites (bold) present in the cytoplasmic domain are also shown. (B) Sequence from clone 54-1 that encodes a stop codon (double underlined] that would truncate thecytoplasmic tail after 16 amino acids (GenBank accession no. U49458). Amino acids encoded by the alternativelyspliced exon are underlined. Sequence from the135-bp alternatively spliced exon insert is shown in uppercase. From www.bloodjournal.org by guest on June 16, 2017. For personal use only. CLONINGCD34 OF A DNA FOR CANINE 1997 Human Canine Murine ~ ~ K ~ ~ --AG""-GGL-""-MQ-H-DT--LLL-WR-V-- ~ ' ' ~ ~ PELPTQGTFS M P R5 0 -------F--TNTETVI-P- TVPTSTEIM" T-TS"-ISp -"-"--H*** * * - N W & $ & - Human Canine Murine EXTRACELLULAR DOMAIN NVSTNVSYQE TTTPSTLGST SLHPVSQHGN EATTNITETT VKFTSTSVIT 100 A--E-T-KR- AI-LTPS-T- TLYS---DSS GT-AT-S--- -HV----E-S-P--E-VE- NI-S-IP--- -HYLIY-DSS KT-PA-S--M -N--V--G-P ~ Human SVYGNTNSSV QSQTSVISTV FTTPANVSTP ETTLKPSLSP GNVSDLSTTS 150 Canine LTp-TM---- -----UI-- SF--T-F--S SV--E---L- --G--ppyp~Murine -GS-TPHTFS -P---PTGIL P--SDSI--S -M-W-S--PS INL-HY-Pm Human TSL*ATSPTK PYTSSSP*** ***ILSDIKA EIKCSGIREV KLTQGICLEQ Canine ---*V----E Y---L--TPSm T P - T - - G ------VK-- -LN-""-L Murine S-FEM----E --AYT-SSA* ****P-A--G - - - - - - - - - - R-A"-"-L 200 250 Human NKTSSCAEFK KDRGEGLARV LCGEEQADAD AGAQVCSLLL AQSEVRPQCL Canine -E----ED-- --m-KLTQ- --EK-p-E-G- - * * - - - - - H- Murine SEA---E--- -EK--D-IQI ---K-E-E-D ---T"---- -------E-- _______ Fig 3. Comparison of the amino acid sequences from the speciesfor which a full-length CD34 sequence has been reported. There is high homology in the cysteine-rich regionof the extracellular domain, the transmembrane domain, and the cytoplasmicdomain, but there is marked divergence of sequence in the amino-terminal end of the molecule.Domains of caCD34 were predicted computer by analysisoftheproteinstructure and the homologyto human and murine proteins. Signal peptide sequences are shaded. Alignments were performed using PILEUP (GCG). I Human Canine Murine LLVLANRTEI SSKLQLMKKH QSDLKKLGIL DFTEQDVASH QSYSQKTLIA 300 ------K--LF-----LR-- ---------R -------G" ----R -____ -M----S--L P------E-- ----R----Q S-NK--IG-- ----R----- H~~~~ Canine Murine LVTSGALLAV LGITGYFLMN RRSWSPTGER LGEDPYYTEN GGGQGYSSGP 3 5 0 TRANSMEMBRANE I CYTOPLASMIC DOMAIN -"--I-"--T"""- - - - - _ _ _ _ -_- - _ _ _ _ _ ___ __ _ _ _ _ _ _ _ - - - - - V - - -1 - -T-- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Human GTSPEAQGKA SVNRGAQKN'G TGQATSRNGH SARQHWADT EL 385 Canine WGP------- -----P-E--- - - - - - - - - - -----M----- - 389 Murine -A-- -T-- - - N-T-- - -E-- - - - - - - - - - - - - - - - - - - - - - - 382 - = identical to human amino acid * = gap in sequence hinge, CH2, and CH3 domains of a murine Ig heavy chain. DNA sequencing verified a correct construct that encoded amino acids 1 through 26 of the human CD34 signal peptide and residues 14 to 293 of the predicted caCD34 sequence fused in frame to the hinge region of murine heavy chain Cy2a.CD34-Ig wasproduced by transient expression in COS cells and purified using immobilizedprotein A. The molecularweight of CD34-Ig determined by SDS-PAGE under nonreducing conditions was -220 kD, with three distinct species being discernable (Fig 4B). Upon reducing conditions, 2 diffuse bands were visible at -90 and 115 kD, consistent with the protein being a homodimer. Because the predictedmolecularweight of CD34-Igwas 50 kD, this suggested that the protein was glycosylated. Treatment of CD34-Ig with N-glycosidase caused a reduction inmolecular weight (data not shown), confirming the presence of Nlinked carbohydrate. Automated N-terminal sequencing was performed to confirm correct processing of the human signal peptide sequence. This produced a sequence ETVITPXTVPTSXEIMXAV (X = unidentified), which is identical to the predicted mature caCD34 mature protein for each ofthe residues identified. The three unidentified residues at positions 7, 13, and 17 are predicted to be T, T, and S, respec- - tively. The failure to identify these residues may be a result of them bearing carbohydrate residues. Production and evaluation of an afJiniry-puriJied rabbit polyclonal antiserum RPacaCD34. Serumfrom arabbit immunized with CD34-Ig was initially tested for reactivity to CD34-Igby ELISA, whichconfirmed an immune response to the fusion protein (data not shown). Subsequent testing of the affinity-purified anti-caCD34 antiserum(RPacaCD34) by Western blotting and ELISA analysis using CD34-Ig and control-Ig showed that an antibody titer at >1:10,000 was present and that greater than 95% of the antibody reactivity was directed at CD34 extracellular domain, with little residual activity against the murine Ig tail (data not shown). In Western blotting studies of leukemic cell lines and cultured endothelial cells, RPacaCD34 recognized a protein of -90 to 100 kD (Fig 5). A similar sized band was found in ML3, 1390, and CLGL 90 cell lines. Endothelial cells expressed aslightly lowermolecular weight form of caCD34, suggesting thepossibility of glycosylation differencesfrom those in the hematopoietic cell lines. In flow cytometry analyses using RPacaCD34, there consistently was positive staining of endothelial cells and the cell lines ML2, ML3, and 1390, which could be blocked by ~ ~ From www.bloodjournal.org by guest on June 16, 2017. For personal use only. McSWEENEY ET AL 1998 97 - . L ) .. " 30 30 21.5 21.5 14.3 14.3 QL" r).r*Ip Fig 4. (A) Chimeric plasmid construct for the expression of the extracellular domain ofcaCD34 used to transfect Cos cells for production of 034-lg.(B) Analysis of CD34-lg by SDS-PAGE and coomassie staining. CD34-lg is expressed as a homodimer and has a molecular weight of 95 t o 100 kD under reducing conditions, whereas the predicted molecular weight is 50 kD. There appears to be more than one isoform being expressed, most likely due t o variable glycosylation. A murine IgG monoclonal isrun as a control. 2 3 4 5 ? . r RpacaCD34 preincubation of antibody with CD34-Ig (Fig 6), but not with a control fusion protein (data not shown), indicating that binding of RPacaCD34 to cell surface epitopes also expressed by CD34-Ig. Endothelial cells, ML3, and 1390 all expressed high-level caCD34, approximately 1 log of fluorescence greater than ML2. MLI and CLGL did not express detectable surface CD34. These results were consistent with PCR-based assays of CD34 mRNA in which strong amplification signals were detected from ML2, ML3, endothelial cells, and 1390 cells, whereas CD34 expression was weak or absent from MLI and CLGL 90 (data not shown). Analysis of bone marrow reproducibly showed that RpacaCD34 stained brightly approximately 1% of cells. These were primarily small mononuclear cells withlow side scatter that were not detected in canine peripheral blood (Fig 7). Cells were only considered CD34+ if bright staining was found as compared with other cells. Some dim staining of larger cells with intermediate side scatter was also observed, and these cells had the light scattering features of monocytes. In preliminary experiments, immunohistochemistryof dog tissues was performed. Positive staining of canine vascular endothelium with little nonspecificbackgroundreactivity was found (Fig 8). To further assess nonspecific staining and whether RPacaCD34 would recognize human endothelial CD34, the human lung was also tested. There was nostaining of human lung tissue, indicating that RPacaCD34 did not contain antibodies that werecrossreactive with humanvascular CD34. Similarly, RpacaCD34 did not stain the human CD34' leukemia cell line KG1 (data not shown). These results were somewhat surprising in view of the shared amino acid homologyseen in the C-terminal end of the extracellular domains of CD34. The staining pattern in canine lung was very similar to that seen withantihuman factor VI11 staining of canine lung andhuman lung (data not -=a = 250 250 = 148 = 97.4 148 97.4 = 60 60 1 6 6 Control Fig 5. Western blotting analysis of cell lines and cultured vascular endothelium using RPcrcaCD34 and control rabbk serum. Whole cell lysates were prepared as described in the Materials and Methods and 15 p L of each was loaded on t o t h egel. Lanes are as follows: 1, CD34l g 10.5 pg); 2, CLGL; 3, 1390; 4, MU; 5, cultured canine endothelial cells. Size markers indicate molecular weight in kilodakons. From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 1999 CLONING OF A DNA FOR CANINE CD34 by cell sorting and assayed for content of CFU-GM. In two separate experiments, CD34' cells were 25- to 50-fold enriched for CFU-GM as compared with CD34""" cells, unfractionatedPBMC, or BMMCthathad undergone a partial lineage depletion (Table I ) . In experiment 2. the result was qualitatively the same. but there was substantially better colony growth, probably due primarily to greater experience usingthepolyclonal antiserum in flow cytometry experiments and to sample to sample variation in progenitor content. DISCUSSION 1390 ML-2 Log Fluorescence Intensity Fig 6. Flow cytometry analysisofCD34expressionon cultured endothelial cells and canine leukemia celllinesusingRpcrcaCD34. Cell lines were stained with RPcrcaCD34 and a second-stage FITCconjugated goat antirabbit polyclonal antibody as described in the Materials and Methods. In each case, stainingwith RPncaCD34 isthe shaded plot, the broken line shows staining with antibody that was preincubated with CD34-lg, and the solid line shows staining with only the second-stage antibody. shown), except that staining wasmore intense with RPacaCD34. Intense staining was noted in high endothelial venules from lymph node and. strikingly, in the capillaries of the myocardium. Progenitor nssuy. To evaluate whether RpacaCD34 recognized canine hematopoietic progenitors, the brightest l % of BMMC that stained with RPacaCD34 were isolated A cDNA library made from the canine leukemia cell line ML2 was used to clone a cDNA for the canine homologue for CD34. The isolated cDNA sequence encoded the mature protein but not the entire signal peptide sequence, and additional S' and 3' cDNA sequence was determined from genomic clones. Attempts to isolate cDNA clones that extended S' of the signal peptide sequence were unsuccessful, suggesting that the RNA molecule had considerable secondary structure. Exon I of caCD34 is extremely GC rich (82.5%), significantly more so than that of exon 1 of the human gene (62%), and this accounted for failures of reverse transcription and difficulty in DNA sequencing of this region. The GCrich sequence including and immediately 5' of the signal peptide sequence canpossiblyform stem-loop-stem structures'" that maybe important in posttranscriptional regulation of protein expre~sion.~" CpG islands found in the first exon maybe important in regulating tissue-specific gene expression by changes in methylation status.MThe genomic structure of caCD34, including the size of the gene (25 kb between exon 1 and the polyadenylation signal), and the proposed introdexon structure are very similar to that of the human" and murine'') CD34 genes. The promoter region is currently being sequenced to identify possible control elements that regulate CD34 expression in dogs, which may be important for subsequent attempts to control gene expression in canine hematopoietic stem cells. This is the third CD34 cDNA cloned, and Genbank and PIR database searches showed no sequences with significant homology to caCD34 with the exception ofhuman CD34 and murine CD34. Because the function of CD34 remains undetermined. it was expected that comparisons of the molecule from the three species would be informative as to the important functional domains of CD34. The predicted protein structure from all three species shows marked homology, with canine and human CD34 showing greater amino acid homology than either does to the murine protein. At the Nterminal endof CD34 there appears to have been only limited evolutionary pressure to conserve specific amino-acid sequence. Extensive 0-linked glycosylation takes place largely on the N-terminal serine and threonine residues of the human CD34 molec~~le.~' Tissue-specific glycosylation differences may affect the tissue-specific functions of CD34, as suggested by the finding that L-selectin binds CD34 to expressed on vascular endothelium but not to CD34 expressed on hematopoietic c e k SThe conservation of the cysteine-rich area in the C-terminal end of the extracellular domain suggests the presence of critical regions for the extracellular functions From www.bloodjournal.org by guest on June 16, 2017. For personal use only. McSWEENEY ET AL 2000 A B "0.00 u e * M 0 0 BM 0.02 PB . . m- . I=100- . . . .... . . . . . . .. .. . . . . . ... .. , . .... ... 1.... . . .:,. .:.. . . . . . . < .......... ..?.=:'.;;:.2y. ..... . . :;. . . ... .,!'., .. .. :. .. .. .. .. . , : . :3 l 50 W e I ::.<r;,;,: M BM ' ' 1.02. 25 PB . .,C,!. , : ;. . ". .i',,..'.' . . . . .. .. .. . .. .. . . .. . . . .. . .. . ....', . ...... .. . . . ..e. . , , . .' .. ' d' I= zoo D C m IS0 100 Side Scatter Side Scatter a50 .. :.. .. .. . .. . .. .. .... . !?,,?...';. . * .::. .! . ... . . . . ,..."ti. . ... ' . .. . . . . . . S. 0 so 0 Side Scatter Side Scatter Fig 7. Flow cytometry of canine bone marrow and peripheral blood showing side scatter versus CD34 (log PE) t o illustrate patterns of staining with RprrcaCD34. Unfractionated bone marrow (C) and peripheral blood (D) were stained with RPacaCD34 followed by a secondstage PE-conjugated polyclonal goat-antirabbit antibody. Red blood cells were lysed with hemolytic buffer. Controls shown for marrow (A) and peripheral blood(B) were thesecond-stage PE-conjugatedgoat-antirabbit antibody. Debris was gated out before analysis based on forward light scatter and side scatter properties of the cells. The percentage of CD34' cells (in boxes) is an arbitrary estimate determined by sfdting a rectangular gate on cells with low side scatter expressing a high level of reactivity t o RPacaCD34. Some nonspecific staining of cells with low-intermediate side scatter is seen, and thesecells have the forwardversus side scatter characteristics. of monocytes (data not shown). of the molecule andlor for maintaining tertiary structure. Cytoplasmic PKC and tyrosine phosphorylation domains may be potentially critical for intracellular signalling functions of CD34. An alternatively spliced transcript of CD34 encodes a premature stop codon that truncates the cytoplasmic domain. Although the size of the alternatively spliced exon differs in dog, human,"* and mouse,43 in each case an identical truncation of the intracellular domain of CD34 is found. The function of the truncated protein is still unknown, although a role in progenitor differentiation associated with loss of signalling functions has been proposed."." In mice, the variant transcript was detected in both hematopoietic and nonhema- topoietic tissues4' and may contribute up to two thirdsof the CD34 mRNA, depending on which tissue is examined. In human CD34' cells, the variant transcript was expressed in relatively higher proportion in the more differentiated CD38' subpopulation as compared with the CD38- subpopulation." In several canine tissues, the varianttranscript comprised less than 10% of the CD34 mRNA, suggesting possible differences to those observed in murine and human tissues. A recombinant fusion protein encoding the extracellular domain of caCD34 wasproduced once thesequenceand reading frame of the cDNA was determined. To generate a disulfide-linked glycosylated molecule, CD34-lgwaspro- From www.bloodjournal.org by guest on June 16, 2017. For personal use only. CLONING OF A DNA FOR CANINE 2001 CD34 I Fig 8. Immunohistochemistry of canine tissues. Sections were prepared and stainedas described in the Materialsand Methods. (A) Canine lung-no primary antibody. (B)Canine lung-RPcrcaCD34. (C) Human lung"RPcrcaCD34. (D) Canine lung-antifactor VIII. (E) Canine lymph node-RPcrcaCD34. (F)Canine myocardium-RPcrcaCD34. (A) through (E)were photographed at low magnification lapproximately 5 0 x ) and (F) was photographed at medium magnification (approximately 130x1. Slides were photographedwith a Leitz Dialux 20 photomicrograph. In (B)and (D), the arrows point t o positive staining of pulmonary capillaries. In (E), the arrow points t o a high endothelial venule. In (F), the arrows pointt o positive staining of vascular endothelium froma myocardial capillary (thin arrow) and from an arteriole (thick arrow). duced in a mammalian cell line. The expression of caCD34 as an Ig fusion molecule facilitated ease of purification using protein A columns. Antibodies specific to the caCD34, isolated by a two-step affinity-binding process, showed relatively little nonspecific staining when used for flow cytometryandwereuseful for initial characterizations of canine hematopoietic cells. However. MoAbs will be required for large-scale cell separation for experimental transplantation studies as well as for more accurate flow cytometry applications. The positive staining of canine vascular endothelium by RpacaCD34 was consistent with patterns of CD34 expression that have previously been reported for human and murine tissuesAS Northern blotting studies in mice'" and humans8 showed that CD34 is expressed in many tissues. The exact cellular origin of these transcripts has not been determined in humans, although murine studies suggest a likely vascular origin in most instance^.^ In humans, patterns of CD34 expression have not been determined using a polyclonal antiserum. Because CD34 epitope differences may From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 2002 McSWEENEY ET AL Table 1. Analysis of CFU-GM From Sorted CD34' Cells REFERENCES Cell No.* Sample CD34' 1 CD34- 1 CD34' 2 CD34- 2 BMMC 2 LIN- 3 LIN' 3 LIN- 4 LIN' 4 LIN' 5 LIN' 5 BMMC 3 BMMC 5 o3 1 3.5 0 63 1 6 0 0 0 0 6 0 ND ND 5 X 103 19 0 1!69 0 9 ND 0 0 0 2 1 ND ND 104 50 0 304t Ot 25t 1 0 2-3 0 ND 1 1 9 Values represent average from duplicate plates. Numbers 1 through 5 refer to dogs used in the experiments. Abbreviations: ND, not done; CD34', sorted caCD34' cells; CD34-, sorted CD34di" cells; L W , lineage-depleted 6°C; LIN', lineage-positive BMMC. * Number of cells plated in experiment. t Results were calculated on basis of plating 5 x IO4 cells. exist between different tissue^,^ the use of a polyclonal antiserum (preferably in conjunction with several MoAbs) may have advantages, and analysis of canine tissues (studies in progress) will give additional information about the expression patterns of CD34. Possible tissue differences in canine CD34 epitopes were suggested by the Western blot findings of different molecular weight isoforms when comparing endothelial cells to several leukemia cell lines. Our primary purpose for cloning caCD34 was to determine whether CD34 could, as in humans, be used as a marker for canine hematopoietic progenitors and, if so, to produce antibodies for studies of canine hematopoiesis. The flow cytometry patterns of caCD34 expression in bone marrow are similar to those observed for CD34 in humans and support the possibility that future canine studies using antibodies to caCD34 will have predictive significance for human studies. The enrichment of CFU-GM in caCD34' BMMC suggests that this marker will also be found on canine hematopoietic stem cells. This will need to be formally proven by showing long-term engraftment after allogeneic transplantation of purified caCD34+ progenitors. The identification of markers for canine hematopoietic progenitors would allow more precise experimental work in the dog in studies that involve exvivo expansion, peripheral blood allografting, cord blood transplantation, and gene therapy. The cloning of the caCD34 gene, as described here, may prove important in this regard. The recent clonings of a canine CD38 homo10gue~~ and the canine interleukin-3 gene (unpublished preliminary results) may soon lead to development of other useful reagents for these studies and thus further strengthen the dog model for use in experimental transplant studies. ACKNOWLEDGMENT We thank B. Larson, H. Childs, and M. Kunz for assistance in preparation of the manuscript. 1. Civin CI, Strauss LC, Brovall C, Fackler MJ, Schwartz JF, Shaper JH: Antigenic analysis of hematopoiesis. 111. A hematopoietic progenitor cell surface antigen defined by a monoclonalantibody raised against KG-la cells. J Immunol 133:157, 1984 2. Katz FE, Tindle R, Sutherland R, Greaves MF: Identification of a membrane glycoprotein associated with haemopoietic progenitor cells. Leuk Res 9:191, 1985 3. Andrews RG, Singer JW, Bemstein ID: Monoclonal antibody 12.8 recognizes a 115-kd molecule present on both unipotent and multipotent hematopoietic colony-forming cells and their precursors. Blood 672342, 1986 4. Fina L, Molgaard HV, Robertson D, BradleyNJ,Monaghan P, Delia D, Sutherland DR, Baker MA, Greaves MF: Expression of the CD34 gene in vascular endothelial cells. Blood 75:2417, 1990 5. Baumhueter S, Dybdal N, Kyle C, Lasky LA: Global vascular expression of murine CD34, a sialomucin-like endothelial ligand for L-selectin. Blood 84:2554, 1994 6. Young PE, Baumhueter S, Lasky LA: The sialomucin CD34 is expressed on hematopoietic cells and blood vessels during murine development. Blood 85:96, 1995 7. Strauss LC, Rowley SD, LaRussa VF, Sharkis SJ, Stuart RK. Civin CI: Antigenic analysis of hematopoiesis. V. Characterization of My-l0 antigen expression by normal lymphohematopoietic progenitor cells. Exp Hematol 14378, 1986 8. Simmons DL, Satterthwaite AB, Tenen DG, Seed B: Molecular cloning of a cDNA encoding CD34, a sialomucin of human hemdtopoietic stem cells. J Immunol 148:267, 1992 9. 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Shaw G , Kamen R: A conserved AU sequence from the 3’ untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46:659, 1986 37. Woodget JR. Gould KL, Hunter T: Substrate specificity of protein kinase C. Use of synthetic peptides corresponding to physiological sites as probes for substrate recognition requirements. Eur J Biochem 161:177, 1986 38. Patchinsky T, Hunter T, Esch FS, Cooper JA, Sefton BM: Analysis of the amino acids surrounding sites of tyrosine phosphorylation. Proc Natl Acad Sci USA 79:973, 1982 39. Devereux J, Haeberli P, Smithies 0: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12387, 1984 40. KozakM: Leader length and secondary structure modulate mRNA function under conditions of stress. Mol Cell Biol 8:2737, 1988 41. Sutherland DR, Watt SM, Dowden G, Karhi K, Baker MA, Greaves MF, Smart JE: Structural and partial amino acid sequence analysis of the human hematopoietic progenitor cell antigen CD34. Leukemia 2:793, 1988 42. Nakamura Y, Komano H, Nakauchi H: Two alternative forms of cDNA encoding CD34. Exp Hematol 21:236, 1993 43. Suda J, Sudo T, Ito M, Ohno N, Yamaguchi Y, Suda T: Two types of murine CD34 mRNA generated by alternative splicing. Blood 79:2288, 1992 44. Fackler MJ, Krause DS, Smith OM, Civin CI, May WS: Fulllength but nottruncated CD34 inhibits hematopoietic cell differentiation of M1 cells. Blood 85:3040, 1995 45. Krause DS, Fackler MJ, Civin CI, May WS: CD34: Structure, biology, and clinical utility. Blood 87:1, 1996 46. Uribe L, Henthorn PS, Grimaldi JC, Somberg R, Hartnett BJ, Felsburg PJ, Weinberg K Cloning and expression of the canine CD38 gene. Blood 86:115a, 1995 (abstr, suppl 1) From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 1996 88: 1992-2003 Canine CD34: cloning of the cDNA and evaluation of an antiserum to recombinant protein PA McSweeney, KA Rouleau, R Storb, L Bolles, PM Wallace, M Beauchamp, L Krizanac-Bengez, P Moore, G Sale, B Sandmaier, T de Revel, FR Appelbaum and RA Nash Updated information and services can be found at: http://www.bloodjournal.org/content/88/6/1992.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. 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