Relationship between Novel Isoforms, Functionally Important Domains, and Subcellular Distribution of CD164/Endolyn*

James Yi-Hsin ChanDagger , Jane E. Lee-PrudhoeDagger , Britt JorgensenDagger , Gudrun Ihrke§, Regis DoyonnasDagger , Andrew C. W. Zannettino, Veronica J. BuckleDagger , Christopher J. WardDagger , Paul J. Simmons||, and Suzanne M. WattDagger **

From the Dagger  Medical Research Council Molecular Hematology Unit, Institute of Molecular Medicine, Oxford OX3 9DS, United Kingdom, the § Wellcome Trust Centre for Molecular Mechanisms in Disease and the Department of Clinical Biochemistry, University of Cambridge, Cambridge CB2 2XY, United Kingdom, the  Hanson Centre for Cancer Research, Institute of Medical and Veterinary Sciences, Adelaide, 5000 South Australia, Australia, and the || Peter McCallum Cancer Institute, Melbourne, 3002 Victoria, Australia

Received for publication, August 31, 2000, and in revised form, October 6, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Functional analyses have indicated that the human CD164 sialomucin may play a key role in hematopoiesis by facilitating the adhesion of human CD34+ cells to the stroma and by negatively regulating CD34+CD38lo/- cell proliferation. We have identified three novel human CD164 variants derived by alternative splicing of bona fide exons from a single genomic transcription unit. The predominant CD164(E1-6) isoform, encoded by six exons, is a type I transmembrane protein containing two extracellular mucin domains (I and II) interrupted by a cysteine-rich non-mucin domain. The 103B2/9E10 and 105A5 epitopes, which specify ligand binding characteristics, are located on the exon 1-encoded mucin domain I. Three human CD164(E1-6) mRNA species, exhibiting differential polyadenylation site usage, are differentially expressed in hematopoietic and non-hematopoietic tissues. This study provides additional evidence that human CD164(E1-6) represents the ortholog of murine MGC-24v and rat endolyn. Comparative analysis of murine MGC-24v/CD164(E1-6) with human CD164(E1-6) revealed two potential splice variants and a similar genomic structure. Whereas the human CD164 gene is located on chromosome 6q21, the mouse gene occurs in a syntenic region on chromosome 10B1-B2. By confocal microscopy, human CD164 in CD34+CD38+ hematopoietic progenitor (KG1B) and epithelial cell lines appears to be localized primarily in endosomes and lysosomes, with low concentrations at the cell surface. However, in a minority of KG1B cells, CD164 is more prominently expressed at the plasma membrane and in the recycling endosomes, suggesting that its distribution is regulated in cells of hematopoietic origin.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The hematopoietic system is composed of a continuum of cells that are hierarchically ordered on the basis of their proliferative and differentiation potentials. At its apex is a rare population of slowly cycling pluripotent hematopoietic stem cells that are not only capable of extensive proliferation or commitment to one of nine lymphoid or myeloid cell lineages, but also have remarkable versatility with their ability to be reprogrammed into non-hematopoietic lineages (reviewed in Refs. 1 and 2). Hematopoietic stem cells and their progeny migrate to and colonize a series of hematopoietic sites during ontogeny. Under steady-state conditions, adult hematopoiesis is restricted mainly to the bone marrow, where hematopoietic and mesenchymal stem cells and their progeny reside in specific microenvironmental niches composed of phenotypically and functionally heterogeneous stromal cells and their associated biosynthetic products, including cytokines, chemokines, and adhesion ligands. Stem cell fate (i.e. migration, homing, cell positioning, differentiation, survival, proliferation, commitment, specific gene expression, reprogramming, and death or apoptosis) is regulated by external cues provided by the associated microenvironmental niche. Recent studies have implicated cooperative interactions between cytokine, chemokine, adhesion, and signaling receptors on hematopoietic stem cells and their progeny as key elements in detecting, translating, and fine-tuning these extrinsic cues (reviewed in Refs. 3 and 4). Despite this, the precise mechanisms of hematopoietic stem cell trafficking and localization within microenvironmental niches in adult and fetal development remain to be determined. The identification of the full complement of receptor/ligand interactions and the molecular mechanisms that determine their involvement in regulating stem cell fate therefore remains one of the major investigative areas in hematopoiesis.

We have recently identified and cloned human CD164, a novel 80-100-kDa sialomucin expressed, although not exclusively, by CD34+ and CD34lo/- hematopoietic stem cells and associated microenvironmental cells (5-12). It contains, in its extracellular region, two mucin domains (I and II) linked by a non-mucin domain, which has been predicted to contain intradisulfide bridges (12). Functional analyses in vitro have indicated that this receptor may play a key role in hematopoiesis by facilitating the adhesion of human CD34+ cells to bone marrow stroma and by negatively regulating CD34+CD38lo/- hematopoietic progenitor cell proliferation (5). These effects involve the CD164 class I and/or II epitopes recognized by the monoclonal antibodies (mAbs)1 105A5 and 103B2/9E10 (9). These epitopes are carbohydrate-dependent and are located on the amino-terminal mucin domain I (6, 7, 9). Recently, Kurosawa et al. (13) and Ihrke et al. (12) demonstrated that murine MGC-24v and rat endolyn share significant sequence similarities with human CD164 (5).

Our aim in this work was to define the size, diversity, and subcellular distribution of the human CD164 family and to compare the structural organization and chromosomal location of the human CD164 gene with its murine counterpart as a first step toward more precisely exploring the functional role of this molecule in vivo in murine model systems and in vitro. Toward this end, we surveyed both human and murine RNAs from many different normal tissues and transformed cell lines for alternatively spliced isoforms. We have further defined the complete genomic structure of these human and murine genes and show that they possess similar genomic structures and similar chromosomal localizations. These results place CD164 within the mucin subgroup that is composed of multiple exons and further demonstrate the diverse chromosomal distribution of this family of molecules. Structural features and subcellular localization studies support the view that murine MGC-24v and rat endolyn genes are the orthologs of human CD164. The new CD164(E1-6) isoform, the major translated product, most closely resembles the described murine and rat cDNAs. The finding of additional novel variants of human CD164 extends the repertoire of the CD164 transmembrane receptor isoforms to four members and suggests that these may differentially interact with cognate ligands to modulate the activity of CD164 in vivo on a variety of cellular targets.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cell Cultures-- The cell lines TF1, KG1A, KG1B, U937, K562, Calu-1, A431, and KATO-III were maintained in RPMI 1640 medium containing 10% (v/v) fetal calf serum. TF1 also required 2 µg/ml recombinant human GM-CSF (R&D Systems, Abingdon, United Kingdom) for growth. Human bone marrow was collected with informed consent and appropriate ethical approval. Human bone marrow stromal reticular cells and CD34+ progenitor subsets were isolated and cultured as described previously (5).

Inhibition of Cell Growth-- The effects of engagement of the CD164 molecule by the CD164 class II mAb 103B2/9E10 on recruitment of cells into the cell cycle were determined in single cell assays as described previously (5). To measure the effects of the CD164 mAbs on nucleated cell production, CD34+ cells (103 cells/culture) were cultured in triplicate in serum-deprived medium containing 10 ng/ml each purified recombinant IL-1beta , IL-3, IL-6, G-CSF, GM-CSF, and Steel factor with 10 µg/ml mAb 103B2/9E10, 105A5, mIgG3, or mIgM. Additional cytokines and mAbs were added on days 7, 14, and 21, and nucleated cells were counted at weekly intervals. To examine the effect of engagement of the CD164 molecule on the growth and development of committed hematopoietic progenitor cells, 561-Dynabead (Dynal, Oslo, Norway)-purified CD34+ cells (103) were incubated with either purified mAb 103B2/9E10 or 105A5 or isotype-matched mIgG3 or mIgM control mAb at concentrations ranging from 0.01 to 30 µg/ml for 1 h at 4 °C. Cells were plated in 0.9% (w/v) methylcellulose (Dow Chemicals, Lake Jackson, TX) supplemented with a 10 ng/ml concentration of each of the recombinant human cytokines IL-1beta , IL-3, IL-6, G-CSF, GM-CSF, Steel factor, and erythropoietin (5). Erythroid burst-forming units (BFU-E) and granulocyte/macrophage colony-forming cells (GM-CFC) were counted at day 14 of culture (5).

CD164 cDNA Probes-- Screening of the human PAC library and subclones and the probing of Southern blots were carried out with the human CD164 cDNA probes h1, h2, h3, and h4, derived from the 105A5 cDNA clone (5, 6) (see the legend to Fig. 1) in the pGEM T vector (Promega, Madison, WI). The murine CD164 cDNA was prepared by PCR amplification of BALB/c mouse heart first strand cDNA (CLONTECH, Palo Alto, CA) using primers mF164 and mCD164-SR or mR164 (see Table I) to the murine MGC-24 cDNA sequence (13) in the DDBJ/GenBankTM/EBI Data Bank (accession number AB028895). Screening of the murine PAC library and subclones and Southern blotting were carried out with the murine CD164 cDNA probes m1 and m2 (described in the legend to Fig. 6A). Each cDNA probe (20-50 ng) was labeled with 50 µCi of [alpha -32P]dCTP (Amersham Pharmacia Biotech, Buckinghamshire, UK) using the T7 Quickprime or Megaprime kit (Amersham Pharmacia Biotech, Uppsala, Sweden) as described previously (6).

Southern Blots of Genomic DNA-- A Southern blot containing human placental DNA digested with EcoRI, HindIII, BamHI, PstI, and BglII was obtained from CLONTECH. Mouse strain 129 genomic DNA (15 µg/digest) was similarly digested and Southern-blotted onto Zeta-Probe membrane (Bio-Rad, Hertfordshire, UK) as described (6). These blots were hybridized with alpha -32P-labeled human CD164 probe h3 or murine probe m1.

Isolation and Subcloning of PAC Clones-- The human PAC library derived from normal human male genomic DNA and the murine PAC (RPC121) library derived from 129/Svev TACfBr mouse spleen genomic DNA in the pCYPAC2N and pPAC4 vectors, respectively, and gridded onto Hybond N filters were provided by the Human Genome Mapping Project Resource Centre (Cambridge, UK). These filters were probed with alpha -32P-labeled human h1 and h2 and murine m1 and m2 CD164 probes as described above. One positive PAC clone containing human CD164 was selected and digested with BamHI into three fragments (B3, B2, and B1 fragments), and the B1 fragment was divided by the BglII restriction site into four fragments (Bgl3.25, Bgl2.61, Bgl7.1, and Bgl4 fragments) and then subcloned into similarly digested pCRScript(SK+) vectors (see Fig. 1). The CD164 gene inserts were sequenced directly using oligonucleotide primers derived from the cDNA and genomic DNA. Alternatively, using pCRScript SK(+) vectors containing the B2 and B3 inserts and the Bgl2.61, Bgl3.25, Bgl4, and Bgl7.1 inserts as templates, nested deletions were generated using the Erase-a-Base technique and the Erase-a-Base® kit (Promega) according to the manufacturer's instructions prior to automatic sequencing using M13 forward or reverse primers. To confirm the junctions between the BamHI and BglII fragments, PCRs on the human PAC clones were carried out using the Expand Long Template system (Roche Molecular Biochemicals, Mannheim, Germany) and the following primer pairs: B3-PCR-F1 and B2-PCR-B1, CD164-2KF2 and MGC-GP-B1, B3.5-R2 and B2.5-F6, B2.5-RF7 and B7-R3, and B7-F2 and B4-FR3 (see Table I). The PCR products were polyethylene glycol-precipitated and sequenced directly using the same primers (6). One mouse PAC clone was selected, digested with BamHI or EcoRI, subcloned into similarly digested pCRScript(SK+) vectors, and PCR-amplified using M13 forward and reverse primers or primers to the cDNA, and the PCR products were sequenced (6).

These sequences and those described below were analyzed using MacVector, Seqed, Assemblign, Analysis, and Sequencher software packages and aligned with each other and with the CD164 cDNAs, and a contig was generated. Sequence data were then analyzed by accessing MacVector and the Prosite (Swiss Institute of Bioinformatics (SIB)), GenBankTM, EMBL, and NCBI Databases for alignment, homology, N-glycan, hydropathy, and motif analyses. O-glycbase on the NCBI site was used for predicting O-glycosylation sites. The signal, transmembrane, and polyadenylation sites were predicted using Signal P (14), TMHMM (15), and WebGene HC-PolyA (16) programs, respectively.

Chromosomal Localization of the Murine CD164 Gene-- Fluorescence in situ hybridization analysis was carried out essentially as described by Buckle and Rack (17). Murine digoxigenin-labeled PAC was co-hybridized with a commercial biotin-labeled mouse chromosome 10 paint (Cambio, Cambridge) to chromosomes prepared from a normal mouse splenic culture stimulated with lipopolysaccharide for 48 h and detected as described (18). Chromosomes were counterstained with 4,6-diamidino-2-phenylindole, and images were captured using a CCD camera and MacProbe Version 4 software (Perceptive Scientific Instruments, Inc., League City, TX).

RNA Preparation-- Total RNAs from the KG1A, K562, U937, TF1, Calu-1, and KATO-III cell lines, and from human bone marrow stromal cells, and from murine strain 129 testis were extracted using the RNAzol or guanidinium isothiocyanate protocol (19, 20).

PCR Analysis of cDNAs-- PCR analyses were carried out with Marathon-Ready cDNAs derived from human bone marrow, placenta, spleen, kidney, and colon or with a murine cDNA panel derived from adult mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis and from day 7, 11, 15, and 17 mouse embryos (CLONTECH) and with appropriate CD164, beta -actin, or GAPDH forward and reverse primers (see Table I) using the Expand High Fidelity PCR system (Roche Molecular Biochemicals) as described by the manufacturer. The products were subcloned into the pMOS-Blue T vector (Amersham Pharmacia Biotech) and sequenced.

RT-PCR Analysis of RNA-- Total human RNAs (2 µg) were reversed-transcribed by standard techniques. PCRs were then carried out as described above on these transcribed cDNAs using CD164 reverse oligonucleotide primers RD1-RD6, MGC-24-B7, MGC-GP-B2, and MGC-GP-B5 (within exons 1-6 and in the 3'-UTR) and the F164 forward primer at the translational start site and GAPDH positive control primers (GAPDH-F and GAPDH-R) (see Table I). The PCR products were subcloned into the pMOS-Blue T vector and automatically sequenced using a 3 pM concentration of the appropriate forward or reverse primer.

3'-Rapid Amplification of cDNA Ends (3'-RACE)-- The 3'-ends of the human and murine CD164 mRNAs were determined by 3'-RACE using Marathon-Ready cDNAs derived from human bone marrow, placenta, spleen, kidney, and colon or from murine testis as templates. The reaction mixtures contained 200 ng of each cDNA sample and a 10 µM concentration of either primer MGC-GP-F2 or MGC-GP-F4 for human or primer mCD164-E5F, mCD164-E6F1, mCD164-3F8, or mCD164-3F10 for mouse and reverse adaptor primer 1 or 2 (see Table I) together with 200 µM each dNTP (Amersham Pharmacia Biotech), Taq/PWO (Pyrococcus woesi) DNA polymerase mixture (Expand Long Template PCR system), and Taq antibody (CLONTECH) in Expand Long buffer using the Expand Long Template PCR system according to the manufacturer's instructions and standard conditions. The PCR products were subcloned into the pMOS-Blue T or pGEM T-easy vector prior to automatic sequencing using the appropriate forward or reverse primers.

Northern Blot Analysis-- Human multiple hematopoietic and non-hematopoietic tissue Northern blots containing poly(A)+ mRNAs were purchased from CLONTECH. The blots were probed with alpha -32P-labeled probes CD164(E1), CD164(E1-4), CD164(E4), CD164(E5), CD164(E6), CD164-PolyA-2, CD164-PolyA-3, and GAPDH, which were generated by PCR amplification as detailed above from the human bone marrow-derived CD164(E1-6) cDNA with the respective primer pairs (see Table I and Fig. 3A): F164 and RD1, F164 and RD4, EXON4-F and RD4, EXON5-F and RD5, CD164(E6)-TM-F and MGC-24-B7, MGC-GP-F4 and MGC-GP-B4, MGC-F7 and CD164-PolyA-3, and GAPDH-F and GAPDH-R. Probes were labeled with [alpha -32P]dCTP using the sequence-tagged site method (21). The human beta -actin (CLONTECH) or GAPDH probe was labeled with [alpha -32P]dCTP using the Megaprime kit. A murine multiple tissue Northern blot containing poly(A)+ mRNAs from murine heart, brain, lung, liver, skeletal muscle, kidney, and testis was probed with [alpha 32-P]dCTP-labeled murine CD164 probe m1 (described in the legend to Fig. 6A). Murine strain 129 testis total RNA was Northern-blotted onto Hybond N membranes (Amersham Pharmacia Biotech) using standard techniques. Using GAPDH (see Table I) and beta -actin probes, similar mRNA levels were found in the different tissues (data not shown), indicating essentially even loading of poly(A)+ RNA in the different lanes. The blots were probed with 32P-labeled murine probe m1 or m3 (see Fig. 6, A and E). The latter was generated by sequence-tagged site labeling of the PCR products from mouse CD164 cDNA using the mCD164-3F10 and mCD164-3R6 primers (see Table I).

RNase Protection-- Two human CD164 probes were constructed for the RNase protection assay. The first probe, PE1, spanning exon 1 plus 390 bases of the 5'-UTR and 83 bases of intron 1, was prepared by digesting the 2.73-kb BamHI fragment of the human PAC clone (B2.73) with the restriction enzymes SalI and SmaI prior to subcloning into the similarly digested pSPT19 vector (Roche Molecular Biochemicals). Probe PE2, containing exons 3-5, was generated by PCR amplification of the CD164 cDNA using the primer pairs FBAM3 and RECO1 (see Table I), followed by insertion into the BamHI/EcoRI-digested pSPT19 vector. The long GAPDH universal control probe (GAPDH-L) was produced by PCR amplification of bone marrow-derived Marathon-Ready cDNA using primer pairs GAPDH-F and GAPDH-R, whereas the short GAPDH universal control probe (GAPDH-S) was produced by PCR amplification of the bone marrow-derived Marathon-Ready cDNA using primer pairs PGF and PGR (see Table I) and inserted into the HindII/HindIII-digested pSPT18 vector. PE1, PE2, and GAPDH plasmids, digested with the KspI, HindIII, and EcoRI restriction enzymes, respectively, for linearization, were labeled with [alpha -32P]GTP (50 µCi; Amersham Pharmacia Biotech) using SP6 RNA polymerase (20 units; Roche Molecular Biochemicals) and the SP6/T7 transcription kit (Roche Molecular Biochemicals) prior to DNase I (20 units) digestion. Total RNAs (30 µg) from Calu-1 and KG1A cells were mixed with the [alpha -32P[GTP-labeled PE1 or PE2 probe or the GAPDH positive control probes (1 × 106 cpm/probe/tube) prior to digestion with ribonucleases A (160 ng/µl) and T1 (2.8 units/µl) (Roche Molecular Biochemicals) and treatment with a 2:1 mixture of 20% (w/v) SDS and 10 mg/ml proteinase K (ICN Pharmaceuticals Ltd., Hampshire, UK) and then electrophoresed on 8% (w/v) polyacrylamide gels (Sequagel, National Diagnostics, Aylesbury, UK) using standard techniques. 32P-Labeled pBR322 MspI-digested DNA (New England Biolabs Inc., Hertfordshire) at 104 cpm was loaded per marker lane. The gels were dried on a Bio-Rad Model 583 gel dryer and exposed to Kodak X-Omat film with intensifying screens at -70 °C.

Indirect Immunofluorescence and Confocal Microscopy-- Calu-1 and A431 cells were grown on glass coverslips. High density suspension cultures of KG1B cells were settled onto poly-D-lysine-coated glass coverslips for 30 min to obtain a dense lawn of attached cells that could be fixed on the coverslips. All three cell types were fixed with 4% (w/v) paraformaldehyde in phosphate-buffered saline for 30 min at room temperature, incubated with 0.25% (w/v) NH4Cl (3 × 5 min), and then blocked and permeabilized with 1% (w/v) bovine serum albumin and 0.1% (w/v) saponin in phosphate-buffered saline for 30 min. Cells were incubated for 1 h simultaneously with two of the following primary antibodies in 0.5% (w/v) bovine serum albumin and 0.025% (w/v) saponin in phosphate-buffered saline: mouse anti-human CD164 mAbs N6B6 and 103B2/9E10 (6), fluorescein isothiocyanate-conjugated mouse anti-human transferrin receptor antibody (Dakopatts, Glostrup, Denmark), rabbit anti-early endosomal antigen-1 antibody (serum 243, a gift from Michael J. Clague) (22), and rabbit antiserum to the cytoplasmic domain of Lamp-1 (a gift from Colin R. Hopkins).2 For primary antibody pairs derived from different species, fluorescein isothiocyanate- or Texas Red-conjugated secondary antibodies to mouse and rabbit IgG (F(ab)2 fragments, developed in donkey; 10 µg/ml; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were used. When both primary antibodies were from mouse, isotype-specific fluorescein isothiocyanate- or Texas Red-conjugated secondary antibodies (F(ab)2 fragments or whole IgG, developed in goat; 10 µg/ml; Southern Biotechnology Associates, Birmingham, AL) were employed. Control cells were labeled with the same two primary antibodies and only one or the other secondary antibody to ensure that no cross-reaction between isotypes was observed. Cells were mounted in Prolong Anti-fade (Molecular Probes, Leiden, The Netherlands). Confocal images were taken with a Leica TCS SP system equipped with a Leitz Plan-Apo 63× objective (NA 1.4) at a pinhole setting of 1 airy unit and a resolution of 1024 × 1024 pixels using a zoom of 2.0-4.0. Adobe Photoshop software was used for image processing.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Complete Sequencing of Human CD164 PAC Clones

To isolate genomic clones, a male peripheral blood leukocyte PAC library was screened with human CD164 cDNA probes h1 and h2. Southern blotting with CD164 probe h3 demonstrated identical patterns of hybridized restriction enzyme-digested fragments from both PAC clones and from human placental genomic DNA, indicating that the PAC clones had not undergone gross rearrangements within the region of the CD164 gene. A single hybridization band was observed with each enzyme, the largest being the BamHI-restricted fragment (B1) of ~17 kb. When the blots were reprobed with cDNA probe h3, two additional BamHI fragments of 2 and 3 kb (B2 and B3, respectively) were identified. These three BamHI fragments span ~21 kb of DNA and contain the whole coding sequence of the human CD164 gene (Fig. 1 and data not shown). Thus, the human CD164 gene comprises six exons that are interspersed with five introns of varying sizes (2.540 kb (intron 1), 1.427 kb (intron 2), 1.788 kb (intron 3), 5.608 kb (intron 4), and 1.494 kb (intron 5)). All predicted splice donor and acceptor sequences matched the G(T/A)G consensus sequence (23). As described previously, this PAC clone was used to localize the human CD164 gene to chromosome 6q21 (6).



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Fig. 1.   Genomic structure of human CD164. Shown is a map of the intron/exon structure of the human CD164 gene. The relationship between the genomic sequence (GenBankTM/EBI accession number AF299340) and the predicted full-length human CD164(E1-6) nucleotide sequence (accession number AF299341) is shown. Human CD164 cDNA probes used to screen PACs and Southern blots were prepared as follows. The SacI/SpeI probe h1 fragment contained bp 1-1907 of cDNA sequence (where bp 1 indicates the translational start site), encompassing the whole coding sequence plus part of the 3'-UTR and part of the pRUF.neo vector. The SpeI/ApaI probe h2 fragment encompassed bp 1908 to 2872 of the 3'-UTR and part of the pRUF.neo vector. Probe h3 comprised a 1.173-kb EcoRV/HindIII CD164 probe from the 105A5 cDNA spanning bp 1309-2487 of untranslated CD164 cDNA sequence in the 3'-UTR. Probe h4 comprised a 612-bp BstXI/NcoI probe derived from bp 42 to 653 of the cDNA.

Identification of Four Splice Variants of Human CD164

Our previous data (5) described the partial sequence of two human CD164 cDNA clones (103B2/9E10 and 105A5) isolated from a human bone marrow stromal cDNA expression library. Complete sequencing of these two cDNA clones revealed cDNA inserts of 2873 bp linked to additional 5'- and 3'-sequences derived from the pRUF.neo vector. Both clones were identical except for six base pair changes, one of which occurred in the coding sequence and resulted in an amino acid change from serine to glycine at position 89 from methionine 1 of the translational start site in the 105A5 cDNA clone (Fig. 2).



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Fig. 2.   cDNA sequence of human CD164. The nucleotide and predicted amino acid sequences of human CD164(E1-6) (GenBankTM/EBI accession number AF299341) are shown. The boundaries for each exon-encoded peptide sequence are indicated (/).The signal cleavage site is marked with a broken arrow; the transmembrane sequence is shown in boldface italic type and is underlined; and the carboxyl-terminal YHTL amino acid motif is boldface and underlined. The presence of cysteines (§) and of predicted N-linked (*) and O-linked (#) glycosylation sites is also shown. The potential amino acids involved in the glycosaminoglycan attachment site are double-underlined. The positions of nucleotides used as primers (MGC-GP-F2, MGC-GP-F4, MGC-GP-B4, and MGC-F7) for 3'-RACE PCR are indicated and listed in Table I. The position of the 3'-UTR deletion that forms the CD164(E1-6)Delta 3'-UTR splice variant is shown in italic type and underlined. The changes to the CD164 sequence that result in the MGC-24 sequence are marked (\). In the 3'-UTR, predicted polyadenylation sites (PolyA-1, PolyA-2, and PolyA-3) are underlined and labeled, whereas the signal sequence is boldface and underlined.

Comparison with the genomic CD164 DNA demonstrates that these human CD164 cDNAs are encoded by exons 1-4 and 6. To identify other splice variants of CD164, cDNAs were amplified from a variety of cell lines and tissues by RT-PCR using the forward primer F164, upstream from and including the ATG codon of the translational start site, and the reverse exon 1-6-specific primer RD1, RD2, RD3 RD4, RD5, or RD6 or the 3'-UTR primer MGC-24-B7, MGC-GP-B2, or MGC-GP-B5 (Table I). The resultant products were Southern-blotted using exon-specific probes (Fig. 3A) and sequenced. For all except the latter four primers, single RT-PCR products corresponding to the appropriate exons were generated with the hematopoietic cell lines TF1, U937, KG1A, and K562; the epithelial cell lines KATO-III and Calu-l; and normal human cultured bone marrow stromal cells, normal bone marrow mononuclear cells, and placental, spleen, kidney, and colon tissues (data not shown). With the exon 6-specific and 3'-UTR reverse primers, two different sequences were obtained in most tissues and cell lines, one containing all exons (CD164(E1-6)) and the other lacking exon 5 (CD164(Delta E5)) (Figs. 2 and 3, B and 3C). In spleen, a third isoform was also found that contained all exons but exon 4 (CD164(Delta E4)) (Figs. 2 and 3, B and C). A fourth splice variant was generated by PCR amplification of spleen and colon cDNAs with the F164 and MGC-GP-B5 primers (Table I). This CD164 variant (Delta 3'-UTR) contained the sequence derived from exons 1 to 6, but lacked 356 bp of the 3'-UTR (Fig. 2). A homologous gene, MGC-24, has previously been identified in the KATO-III cell line (24). The sequence for this cDNA is identical to our cDNA sequence from bp 1 to 523, but then splices onto the 3'-UTR from bp 1120 to the TGA stop codon at bp 1164-1166. This would splice within the predicted transmembrane region and create a new 3'-amino acid sequence (Fig. 2). Considerable efforts were made to identify the MGC-24 transcript by RT-PCR using F164 and the reverse primer MGC-GP-B2 (Table I) in our tissues and cell lines, including KATO-III, but these proved unsuccessful.


                              
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Table I
Oligonucleotide sequences and PCR primers
bp 1 occurs at the translational start site. Primers preceded with "m" refer to those used for murine CD164 analysis. NA, not applicable.



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Fig. 3.   Splice variants of human CD164 mRNAs. A, shown are the exon-specific sequences derived from human CD164(E1-6) cDNA and used for probing Northern blots. The three polyadenylation sites (PolyA-1, PolyA-2, and PolyA-3) are labeled. B and C, RT-PCR products from human non-hematopoietic and hematopoietic tissues, respectively, and cell lines were generated using the F164 and MGC-GP-B2 primers and analyzed by agarose gel electrophoresis and ethidium bromide staining (upper panel) or Southern-blotted and probed using CD164(E5)-specific (middle panel) and CD164(E4)-specific (lower panel) probes. The cell lines and tissues used are indicated above the gels. Lanes -ve lacked DNA template. Lanes m contained a 1- kb DNA molecular size ladder (Life Technologies, Inc.).

Our recent studies (8) have shown that the human CD164 protein is widely expressed in both hematopoietic and non-hematopoietic tissues. To test whether the predominant splice variant of human CD164 contained exons 4 and 5 as in CD164(E1-6), total RNAs from the colonic epithelial cell line Calu-1 (Fig. 4B) and from the hematopoietic cell line KG1A (Fig. 4D) were analyzed by an RNase protection assay. The relative levels of the different transcripts were analyzed using probe PE1 to protect exon 1 and probe PE2 to protect exons 3-5 of human CD164 (Fig. 4, A and C). The results in Fig. 4 (B and D) clearly demonstrate major protected fragments of 109 and 171 bases for exon 1 and exons 3-5, respectively. This indicates that CD164(E1-6) is the major isoform in these cell lines.



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Fig. 4.   RNase protection analyses reveal that human CD164(E1-6) is the predominant isoform in Calu-1 and KG1A cells. A and C, the 32P-labeled CD164-PE1, CD164-PE2, and short and long GAPDH (GAPDH-S and GAPDH-L, respectively) control probes were hybridized with 30 µg of total RNA from the Calu-1 and KG1A cell lines, respectively, prior to treatment with RNases A and T1, and electrophoresis on 8% polyacrylamide gels. B and D, a PE1-protected RNA fragment of 109 bp, a PE2-protected RNA fragment of 171 bp, a GAPDH-S-protected RNA fragment of 50 bp, and a GAPDH-L-protected RNA fragment of 180 bp are indicated. Lanes m contained a 32P-labeled pBR322 MspI-digested DNA marker. Arrows indicate the positions of the unprotected probes in A and C and the protected sequences in B and D.

Northern Blot and 3'-RACE PCR Analyses Reveal Three Species of Human CD164 mRNA with Different Polyadenylation Sites

Composite poly(A)+ RNA Northern blots comprising multiple hematopoietic (Fig. 5, A and C) and non-hematopoietic (Fig. 5, B and D) tissues were analyzed using exon- and 3'-UTR-specific cDNA probes (Fig. 3A). Similar results were obtained whether probes to exons 1-4 (Fig. 5, A and B, respectively) or to exon 1, exon 4, exon 5, or exon 6 were used (data not shown), confirming human CD164(E1-6) as the major isoform in the tissues and cell lines examined. All the tissues expressed at least two bands of ~3.2 and 2.8 kb, except brain, which appeared to express the 3.2-kb mRNA only (Fig. 5B). Although the 3.2-kb band appeared to be the most prominent mRNA species in all tissues, both the 3.2- and 2.8-kb species were expressed in slightly variable amounts in the different hematopoietic tissues examined. The 2.8-kb band was more prominent in spleen, lymph node, peripheral blood leukocytes, and fetal liver, but little was detected in thymus and bone marrow (Fig. 5A). The variability in mRNA expression was more evident on the non-hematopoietic blot, with the most prominent bands occurring in heart and placenta (Fig. 5B). Moreover, some tissues expressed another band of 1.2 kb (Fig. 5B). This band was most obvious in placenta, but was also visible in pancreas after short autoradiographic exposures. However, after long exposures, it became evident in hematopoietic tissues and adult liver and skeletal muscle.



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Fig. 5.   Northern blot analyses identify three CD164 mRNA species with different polyadenylation sites. A and C, poly(A)+ RNA Northern blots from human multiple hematopoietic tissues: spleen, lymph node, thymus, peripheral blood (PB) leukocytes, bone marrow, and fetal liver. B and D, poly(A)+ RNA Northern blots from multiple non-hematopoietic tissues: heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. Blots were probed with the CD164(E1-4) (A and B) and CD164-PolyA-3 (C and D) probes indicated in Fig. 3A. The RNA ladder (0.24-9.5 kb) from Life Technologies, Inc. was used as the molecular size marker.

These Northern analyses suggested that variability in mRNA size was due to usage of different polyadenylation sites. This was confirmed by 3'-RACE PCR of cDNAs derived from bone marrow, placenta, spleen, kidney, and colon using the MGC-GP-F2 or MGC-GP-F4 forward primer (Fig. 2 and Table I) and adaptor 1 as the reverse primer (Table I). Two PCR products of ~700 and 1500 bp were generated with the MGC-GP-F4 primer, and one of 600 bp with the MGC-GP-F2 primer. Sequencing confirmed that the human CD164 mRNAs terminated at three different polyadenylation sites, PolyA-1, PolyA-2, and PolyA-3, in human CD164 mRNA (Fig. 2). Computer analysis of these sequences using the WebGene polyadenylation site program indicated the existence of three signal sites associated with these three polyadenylation sites (Fig. 2). The first and second signal sites (25, 26), which serve as the binding site for the 160-kDa protein cleavage polyadenylation specificity factor, are AUUAAA, and the third signal site for the cleavage polyadenylation specificity factor is AAUAAA. The U(T)-rich elements, which serve as the binding site for the 64-kDa cleavage stimulation factor protein CstF (25, 26), were also identified 10-30 bp downstream from the first and second polyadenylation sites. All three polyadenylation sites were confirmed by hybridizing Northern blots with PCR probes between PolyA-1 and PolyA-2 and between PolyA-2 and PolyA-3 (Fig. 3A). One band (3.2 kb) was identified with the CD164-PolyA-3 probe (Fig. 5, C and D), and two bands (3.2 and 2.8 kb) with the CD164-PolyA-2 probe; and in tissues such as placenta, all three RNA species were detected using the exon 1-4- and exon 5-specific probes (Fig. 5B and data not shown).

Conservation of the Genomic Structure, Chromosomal Localization, and Differential Polyadenylated mRNAs of Murine CD164

For comparison, the murine CD164 cDNA was isolated by PCR amplification from a murine heart cDNA library and found to be essentially identical to that described for murine MGC-24v by Kurosawa et al. (13) except for two base pair changes at positions 72 and 73 from the ATG codon of the translational start site. However, these base pair changes did not alter the predicted amino acid sequence and may represent polymorphic variation between mouse strains. Sequence analysis of the murine CD164 PAC clone 488F16 revealed an intron/exon structure identical to the human gene (Fig. 6, A and B). PCR analysis of murine adult and embryonic tissues and sequencing of the PCR products revealed that murine CD164(E1-6) was the major isoform detected with only one variant, CD164(Delta E3), lacking exon 3, which was found in a few cases in adult heart (data not shown). Comparative analyses of murine tissue Northern blots probed with murine CD164 probe m1 (Fig. 6A) revealed a band at ~3.2 kb in the tissues examined, viz. heart, brain, lung, liver, skeletal muscle, kidney, and testis (Fig. 6C). This band was particularly prominent in liver, kidney, and testis. The 2.8-kb mRNA species was not evident on the murine Northern blot. However, it was of interest to note that in murine testis, the 1.2-kb band was also detected as a major mRNA species (Fig. 6, C and D). As with the human cDNA, 3'-RACE PCR of cDNA from murine testis and Northern blot analyses identified the 3.2- and 1.2-kb bands as RNA species with different polyadenylation sites (Fig. 6 (D and E) and data not shown). The murine PAC 488F16 clone was used to localize the murine CD164 gene to chromosome 10B1-B2 (Fig. 7), a region syntenic with human chromosome 6q21.



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Fig. 6.   Genomic structure of murine CD164. A, shown are the murine CD164 cDNA probes. Murine probe m1 contained bp 1-622 of cDNA sequence, with the ATG codon at bp 1-3 as the translational start site. Probe m2 encompassed bp 939-2091 of the 3'-UTR. B, shown is a map of the intron/exon structure of the murine CD164 gene. The relationship between the genomic sequence (GenBankTM/EBI accession number AF299344) and the predicted full-length murine CD164(E1-6) nucleotide sequence (accession number AF299345) is shown. C, shown is a Northern blot of murine tissues containing poly(A)+ mRNAs from heart, brain, lung, liver, skeletal muscle, kidney, and testis probed with probe m1. D, shown is a Northern blot of murine testis mRNA probed with probes m1 and m3 (see A and E). The RNA ladder (0.24-9.5 kb) from Life Technologies, Inc. was used as the molecular size marker. E, the potential polyadenylation sites (PolyA-1 and PolyA-2) in the 3'-UTR of murine CD164(E1-6) identified by 3'-RACE are underlined and labeled, and their signal sequences are in boldface and underlined. Nucleotides used to design primers mCD164-3F10 and mCD164-3R6 for PCR amplification of the murine probe m3 are shown in italic type and are underlined. Numbers on the right indicate the base pair position. bp 1 designates the first base pair of the predicted translational start site.



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Fig. 7.   Chromosomal localization of murine CD164. A, shown is a fluorescence in situ hybridization image of the PAC 488F16 signal (arrowed) co-localized with the mouse chromosome 10 paint on a normal mouse chromosome spread. B, the PAC signal (arrowed) is localized on mouse chromosome 10 to bands B1-B2 by comparison with a mouse chromosome 10 ideogram.

Functionally Important Domains and Motifs of CD164

Protein Structure-- With the intention of predicting functionally important structural features of CD164, we compared the conserved characteristics within the exon-defined amino acid sequence of human CD164(E1-6) with those of murine CD164(E1-6) and the predicted sequence of rat endolyn/CD164(E1-6) (12) (Fig. 8).



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Fig. 8.   Amino acid similarities and structures of CD164 from different species. A, amino acid similarities of CD164 from human, mouse, and rat determined using the ClustalW program from MacVector software. Amino acid identities (dark gray) and similarities (light gray) are indicated. The exons (E1-5), O-linked (triangle ) and N-linked (black-down-triangle ) carbohydrates, and cysteine residues (§) are indicated for human CD164(E1-6) (hCD164(E1-6)) above the sequence and for murine CD164(E1-6) (mCD164(E1-6)) below the sequence. TSS, translational start site. The signal cleavage sites are indicated by bent arrows, and the transmembrane (TM) sequence by a double-headed arrows. B and C, diagrams for human and murine CD164(E1-6), respectively, highlighting mucin domains I and II; the regions defined by exons 4-6; and the positions of class I-III epitopes recognized by human CD164 mAbs 105A5, 103B2/9E10, and N6B6, respectively (9). The diagrams are based on the predicted structure of rat endolyn (rendolyn) (12).

The highest amino acid identities occurred among the exon 6-encoded peptides, with 94.5 and 96.4% identities for the respective mouse and rat sequences compared with the human sequence. This high degree of homology was mainly due to the completely conserved transmembrane and cytoplasmic domains encoded by this exon. The carboxyl-terminal amino acids have been suggested to constitute a targeting motif of the type YXXø (with X being any amino acid and ø being a bulky hydrophobic amino acid) interacting with clathrin-adaptor complexes involved in vesicle traffic (12). Lower amino acid identities were observed for peptides encoded by exons 1 (50 and 44.8%), 2 (both 57.1%), 3 (41.7 and 37.5%), 4 (both 69.2%), and exon 5 (73.7 and 52.6%). The non-mucin domain encoded by exons 2 and 3 contains, in all these species, eight cysteines, four of which are predicted to be involved in the formation of intramolecular disulfide bridges (12). This domain links mucin domains I and II, which are encoded by exon 1 and exons 4 and 5 and part of exon 6, respectively. Using the Prosite scan protein analysis program, we confirmed the presence of sequences CFNVSVVNTTCFW, CVNATFTNNITCFW, and CVNATLTNNITCVW in the exon 2-encoded non-mucin domains of human, mouse, and rat CD164, respectively (12). These sequences belong to the consensus sequence C(LVFYR)X7,8(STIVDN)CXW, one of two cytokine receptor consensus motifs found in such molecules as the erythropoietin, G-CSF, GM-CSF, IL-4, IL-6, and thrombopoietin receptors (reviewed in Ref. 27). Both murine and human CD164(E1-6) possess 32 potential O-linked and 9 N-linked oligosaccharide attachment sites, whereas rat endolyn/CD164(E1-6) is predicted to contain 35 O-linked and 8 N-linked glycosylation sites. Some important characteristics of the predicted exon-defined domains of human CD164 isoforms are summarized in Table II. The similarities in the structures of human and murine CD164(E1-6) are illustrated diagrammatically in Fig. 8 (B and C).


                              
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Table II
Summary of the characteristics of predicted functional extracellular domains of human CD164

Functional Importance of Mucin Domain I Epitopes-- Our previous data indicated that CD164 is present at the surface of cells, where it may function as an adhesion molecule for attachment of CD34+ hematopoietic progenitor cells to the stroma and as a negative regulator of CD34+CD38lo/- hematopoietic progenitor cell proliferation (5). The class I and II epitopes of human CD164 defined by mAbs 105A5 and 103B2/9E10, respectively (9), mediate these functional effects. mAb 105A5 identifies a sialic acid epitope on O-linked oligosaccharides, whereas mAb 103B2/9E10 identifies an N-linked oligosaccharide-containing epitope, both on mucin domain I (9) (Fig. 8B). Previous single cell analyses using mAb 103B2/9E10 as an inhibitor of proliferation (5) demonstrated that this mAb inhibits the recruitment of single CD34+CD38lo/- cells, the more primitive hematopoietic precursors, into the cell cycle in serum-deprived media in the presence of the cytokines IL-6, IL-3, G-CSF, and Steel factor. We have extended these experiments in Fig. 9 to further demonstrate the importance of mucin domain I. Both mAbs 105A5 and 103B2/9E10 prevented the generation of more mature nucleated cells from CD34+ cells over 4 weeks of culture in serum-deprived liquid medium containing cytokines (Fig. 9A). That this was most likely due to inhibition of recruitment of CD34+CD38lo/- cells into the cell cycle was further substantiated by the finding that the inhibition included nucleated cells from both the erythroid and myeloid lineages (Fig. 9, B-E). The growth of day 14 GM-CFC (Fig. 9, B and C) as well as BFU-E (Fig. 9, D and E) precursors from CD34+ bone marrow cells was inhibited in a dose-dependent manner when clonogenic methylcellulose cultures containing cytokines were grown after preincubation of the cells with the class I and II CD164 mAbs.



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Fig. 9.   CD164 antibodies inhibit hematopoietic progenitor cell proliferation. A, shown is the inhibition of nucleated cell production when CD34+ cells (103 cells/culture) were grown in serum-deprived medium containing IL-1beta , IL-3, IL-6, G-CSF, GM-CSF, and Steel factor with a 10 µg/ml concentration of CD164 mAb 103B2/9E10 or 105A5 or isotype-matched mIgG3 or mIgM mAb. Results are means ± S.E. (n = 3) of the nucleated cells generated at days 0, 14, 21, and 28 of culture. B-E, 561-Dynabead-purified CD34+ cells (103 cells/culture) were incubated with 0.01-30 µg/ml concentrations of mAb 103B2/9E10 or mIgG3 (B and D) or mAb 105A5 or mIgM (C and E) for 1 h at 4 °C and then grown in methylcellulose cultures as described under "Experimental Procedures." The production of GM-CFC (B and C) and BFU-E (D and E) was scored at day 14 and is expressed as colony-forming cell frequencies. Results are means ± S.E. (n = 3) of colony numbers/103 cells plated.

Subcellular Distribution of CD164-- We have demonstrated previously (6, 8) that CD164 is more highly expressed on the surface of primitive CD34+CD38lo/- cells than on the surface of more mature CD34+CD38+ hematopoietic progenitor cells. Furthermore, our preliminary results indicate that, during differentiation, the majority of CD164 is lost from the cell surface and becomes distributed inside CD34+CD38+ cells.3 In non-hematopoietic cells, rat endolyn/CD164(E1-6) is primarily localized in endosomal and lysosomal compartments (12, 28, 29). Thus, we examined in greater detail the intracellular localization of human CD164 in epithelium-derived cell lines (Calu-1 and A431) and in the CD34+CD38+ hematopoietic cell line KG1B by confocal microscopy using the class II mAb 103B2/9E10 (see above and Fig. 8B) or the class III mAb N6B6, which preferentially recognizes the peptide backbone encoded by exons 2 and 3 of human CD164 (Fig. 8B). Both antibodies generally gave a very similar labeling pattern in all three cell lines examined (data not shown). In all cases, the majority of CD164 was found in structures inside the cells (Fig. 10, left panels).



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Fig. 10.   Subcellular distribution of CD164 revealed by confocal microscopy. Calu-1 (A-F) and KG1B (G-K) cells were fixed and permeabilized and then double-labeled with one of two antibodies to CD164 (N6B6 and 103B2/9E10 as indicated (left panels)) and an antibody to a marker of early recycling endosomes (transferrin receptor (TfR); B and H), early endosomes (early endosomal antigen-1 (EEA1); D), or lysosomes (Lamp-1; F and K). White arrows on the corresponding left and right panels point to structures that are positive for two markers; pairs consisting of a white and a black arrowhead on the corresponding left and right panels indicate structures in which only one of the antigens is present. Note that in A and B, white arrows indicate the only two structures in this field in which CD164 and the transferrin receptor appear to coincide. In all other cases, the arrows point to just a few of many examples with overlapping label. The images in G and H are montages of four selected fields (two for each CD164 mAb shown) with cells in which CD164 is seen at the cell surface and in recycling endosomes. Cells in I and K show the average distribution of CD164 and Lamp-1 in KG1B cells. Note that one cell in the field is negative for the 103B2/9E10 epitope (black arrowhead). No such negative cells were seen with mAb N6B6. The bar represents 20 µm for A-F and 10 µm for G-K.

Cell-surface label was also observed, but, in fixed and permeabilized cells, was clearly less pronounced than the intracellular label. Interestingly, in agreement with previous data (8), surface CD164 was somewhat easier to detect with mAb N6B6 than with mAb 103B2/9E10. In general, the N6B6 antibody gave a slightly more homogeneous pattern with regard to the labeling intensity of CD164-positive structures within a single cell and also among cells. This was especially obvious in KG1B cells, where a minority of cells did not appear to express the 103B2/9E10 epitope or expressed it only very weakly, whereas the level of staining with mAb N6B6 was not significantly reduced. This indicated that CD164 molecules carrying the 103B2/9E10 epitope were down-regulated in a subset of KG1B cells. The common labeling pattern in all three cell lines was consistent with CD164 being primarily located in endocytic compartments. Immunoreactivity was found in punctate structures of various sizes that were located throughout the cells and that were slightly more concentrated in the perinuclear area. Antibodies to various known marker proteins for endocytic compartments were used in double labeling experiments in conjunction with mAbs N6B6 and 103B2/9E10 to confirm and to define the distribution of CD164 throughout the endocytic pathway. A good coincidence of CD164 and the lysosomal marker Lamp-1 was found in all three cell lines (Fig. 10, E/F and I/K, compare white arrows). In both Calu-1 and A431 cells, there were also significant numbers of strongly CD164-positive structures that were Lamp-1-negative (Fig. 10, E and F, black arrowheads; A431 cells not shown). These structures were identified as an early endosomal population by the presence of the marker early endosomal antigen-1 (Fig. 10, C and D), but did not appear to be recycling endosomes since they did not contain significant amounts of the transferrin receptor (data not shown). This receptor was seen in smaller punctate structures, which were enriched in two locations as expected, namely, in a distinct perinuclear region and underneath or at the plasma membrane (Fig. 10B). Although some CD164 was also seen in small structures similar in size and number to those containing the transferrin receptor, both populations were clearly distinct from each other. In the majority of KG1B cells, CD164 was seen primarily in the same large structures as Lamp-1 (Fig. 10, I and K), although there was some overlap with early endosomal antigen-1 (data not shown). Interestingly, in a minority of KG1B cells, CD164 was found more prominently at the cell surface or in smaller vesicular structures that were positive for the transferrin receptor. This suggested that, in these cells, a significant fraction of CD164 molecules were recycling between the cell surface and early endosomes (Fig. 10, G and H). In a few of these cells, there was almost no CD164 label seen in other intracellular structures than transferrin receptor-positive endosomes, whereas other cells contained simultaneously varying amounts of label in larger structures typical of lysosomes. Altogether, these microscopy results show that, in many hematopoietic and non-hematopoietic cell lines, only a small amount of CD164 is localized at the cell surface, whereas the majority is distributed over endosomes and lysosomes. However, the variation seen among KG1B cells, i.e. the spectrum from almost entirely lysosomal to almost exclusively cell-surface and early and recycling endosomes, indicates that a mechanism exists by which the localization of CD164 can be regulated.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The identification of cell-surface receptors that regulate hematopoietic stem cell fate is critical to our understanding of the molecular events that control the plasticity of such cells, their proliferative capacity, their migration to the correct microenvironmental niches, and their differentiation into one of nine hematopoietic lineages. In turn, such knowledge is invaluable for clinical applications relating to stem cell usage. Recently, we have demonstrated that one such cell-surface receptor, CD164 (reviewed in Ref. 7), potentially plays a key role in regulating the recruitment of primitive CD34+CD38lo/- hematopoietic stem/progenitor cells into the cell cycle (8). In this study, we have further substantiated that the two glycosylation-dependent class I and class II epitopes present on the exon 1-encoded domain are important for mediating the inhibition of CD34+CD38lo/- hematopoietic progenitor cell proliferation by preventing the development of erythroid and myeloid lineages and the subsequent generation of differentiated nucleated cells in vitro. Both these and other recent (5, 30-33) studies indicate that CD164 as well as other hematopoietic sialomucins such as CD34, CD43, and P-selectin glycoprotein ligand-1 function as a group of negative regulators by preventing hematopoietic stem/progenitor cell proliferation and differentiation.

Moreover, in this work, we report the genomic cloning of human CD164. This human CD164 gene spans at least 22 kb of DNA and contains six exons. Although all the sialomucins mentioned above have similar structural and functional characteristics, their genomic structures and chromosomal localization diverge from each other to varying degrees. For molecules such as human CD43 and P-selectin glycoprotein ligand-1, which contain mucin domains only, the cDNA coding sequence is specified by a single exon. The CD43 and P-selectin glycoprotein ligand-1 genes encompass 4.6 and 11 kb of DNA, respectively, with each containing a single intron that interrupts the sequence specifying the 5'-untranslated region of the mRNAs. These genes are located on chromosomes 16p11.2 and 12q24, respectively (reviewed in Ref. 7). In contrast, genes such as CD34 that contain Ig-like or non-mucin cysteine-rich domains are composed of multiple exons. The human CD34 gene, for example, spans at least 26 kb of DNA, contains eight exons, and is situated on human chromosome 1q32. Thus, CD164 belongs to that subgroup of mucins that are composed of multiple exons, with human CD164 mapping to chromosome 6q21 and murine CD164 to the syntenic chromosome 10B1-B2. This supports the view that the function of molecules such as P-selectin glycoprotein ligand-1 is regulated primarily through post-translational modifications, whereas that of molecules such as CD164 with multiple exons may have more sophisticated regulatory mechanisms, which involve not only post-translational modifications of the oligosaccharide side chains, but also differential exon usage. The complete distribution and functional relevance of the different CD164 splice variants await further analysis.

As a prelude to generating CD164 knockout mice for functional studies in vivo, we have also completely sequenced the putative mouse ortholog of human CD164 and demonstrated a similar distribution of exons. Although differences in the intron and exon sizes are seen between the mouse and human genes, the predicted proteins are similar in size and structure. Furthermore, we have identified two and three isoforms differing in coding sequences for murine and human CD164, respectively. The longest isoform, CD164(E1-6), which is encoded by all six exons, appears to predominate in the tissues and cell lines analyzed. This is in contrast to the originally cloned human CD164 or CD164(Delta E5), which lacks the exon 5-encoded domain. An additional transcript was detected in human CD164 using RT-PCR amplification. This contained all six exons, but lacked 356 bp of the 3'-UTR. In agreement with the recent studies by Kurosawa et al. (13), the predominant mouse isoform is encoded by all six exons. A soluble isoform of human CD164 termed MGC-24 has been described previously (24). Despite extensive RT-PCR analyses, we were unable to detect this soluble variant. From analysis of our genomic sequence, this variant would be derived from exons 1 to 5 and part of exon 6, which would be spliced within the transmembrane region onto a sequence within the 3'-UTR.

On Northern blots, three human and two mouse CD164(E1-6) mRNAs containing different polyadenylation sites were detected, with the shortest transcript of ~1.2 kb most highly expressed in placenta and testis, respectively. The importance of these short transcripts remains to be determined. Interestingly, in a review of 33 genes, Edwalds-Gilbert et al. (26) identified the testis as a "hot spot" for differential polyadenylation site usage. CD164 is an example of a gene whose expression depends on differential usage of polyadenylation sites within a single 3'-UTR. The conserved distribution of the 3.2- and 1.2-kb CD164 transcripts between mouse and human suggests (i) that a mechanism may exist to regulate this tissue-specific polyadenylation and (ii) that differences in polyadenylation are important for the expression and function of CD164 in different tissues. Other research has suggested that genes containing multiple tandemly arrayed polyadenylation sites will select the most 5'-site preferentially. This does not seem to be the case in our studies, where the longest transcript was prevalent in all tissues. Furthermore, studies suggest that the 3'-UTR regulates both the stability and translatability of the mRNA transcript (reviewed in Refs. 34 and 35) as well as controls its nuclear export and subcellular targeting (36). In the human and murine 3.2-kb CD164 mRNA transcripts, there are 15 and 7 AUUUA sequences, respectively. These cis-acting AU-rich elements are thought to be destabilizing elements in such genes as GM-CSF, c-fos, and interferon-beta (reviewed in Refs. 26 and 36), although the optimal destabilizing sequence (UUAUUUAU) is not found in either mouse or human CD164. The choice of polyadenylation sites also depends on the activities and amounts of tissue-specific polyadenylation factors. Alternative polyadenylation usage, if it affects the stability and translatability of mRNA transcripts, would also affect the amount of protein transcribed and thus be of functional relevance. For example, eukaryotic initiation factor-2alpha contains multiple 3'-UTR polyadenylation sites. Two of the mRNA species of 1.6 and 4.2 kb that are transcribed from this gene in activated T cells show differences in stability and translatability. The 1.6-kb transcript is less stable, but more readily translated in vitro. Thus, an increase in polyadenylation enzyme activity as T cells move from the G0 to S phase of the cell cycle leads to a shift to the shorter transcript and a concomitant increase in protein production (reviewed in Ref. 26). A similar scenario may apply for CD164. However, situations in which the short transcript is up-regulated are likely to be underrepresented in the "steady-state" blots used in this study.

In addition to similarities in the long and short mRNA transcript sizes and the predominance of the CD164(E1-6) isoforms in both mouse and human, our studies provide further evidence to confirm that the murine gene described here is the ortholog of human CD164. This is evidenced by the highly conserved amino acid similarities and protein structure as a type I integral transmembrane sialomucin containing two extracellular mucin domains linked by a non-mucin domain as well as the conserved chromosomal localization. Thus, murine CD164 provides an ideal model for the function of the human CD164 gene in vivo in mouse gene knockout/knockin studies. This is particularly important if the effects of CD164 engagement by its cognate or surrogate ligands on the proliferation and adhesion of hematopoietic stem/progenitor cells in vitro demonstrated in this and previous (5) studies are to be reproduced in vivo.

Two other aspects of the structure of CD164 are of particular interest with respect to putative regulatory events. First, CD164 shares one of several conserved features of a cytokine-binding pocket (12). In this respect, it is notable that evidence exists for a class of cell-surface sialomucin modulators that directly interact with growth factor receptors to regulate their response to physiological ligands. One such example is the membrane-bound ASGP2 subunit of rat sialomucin MUC4 (37). This molecule interacts in cis through its epidermal growth factor-1 domain with the extracellular region of ErbB2, a cell-surface tyrosine kinase receptor implicated in embryonic cardiac and neural tissue development. ErbB2 does not appear to bind diffusible ligands directly, but instead acts as an auxiliary co-receptor to enhance signaling via the ErbB receptor network. Growth factor ligands such as neuregulin-1beta bind to the related epidermal growth factor receptor to promote heterodimerization with ErbB2 and consequently to potentiate activation and signaling of the ErbB2 tyrosine kinase. Interactions of the sialomucin ASGP2 subunit with ErbB2 are thought to increase the degree of activation of ErbB receptors or the number of receptors available for activation (37). This in turn would regulate the specificity and strength of receptor signaling. In the case of CD164, the cytokine receptor motif in its non-mucin domain is reminiscent of the non-mucin ASGP2 subunit of the MUC4 sialomucin, although it is unclear if CD164 contributes to cytokine binding or signaling. However, we are now addressing this question.

Second, the cytoplasmic tail of CD164 contains a carboxyl-terminal YHTL motif of the type YXXø found in many endocytic membrane proteins or receptors (12, 38, 39). These tyrosine-based motifs bind to the µ-subunit of adaptor-protein complexes, which mediate the sorting of membrane proteins into transport vesicles from the plasma membrane to the endosomes and between intracellular compartments (38, 39). The cytoplasmic tail of rat endolyn/CD164(E1-6) has been shown to direct the targeting of the protein to endosomes and lysosomes in non-hematopoietic cells (12). Here, we have demonstrated that human CD164 is not always primarily located in late endocytic compartments. Although this was the usual distribution found in human epithelial cell lines and in the majority of KG1B cells used in this study, CD164 was more prominently expressed at the cell surface and in recycling endosomes in a minority of KG1B cells. The KG1B cell line is considered equivalent to CD34+CD38+ hematopoietic progenitor cells. Our earlier studies showed that CD164 is more highly expressed at the cell surface of primitive CD34+CD38lo/- progenitor cells than on the more mature CD34+CD38+ progenitors (6, 8). The current results are consistent with the notion that a minority of KG1B cells have a more primitive phenotype. It may be that CD164 present at the cell surface binds its cognate ligand, which is internalized together with its receptor into endocytic compartments, where it is possibly released while CD164 returns via recycling endosomes back to the cell surface. The distribution of CD164 in the majority of KG1B cells indicates that, in later stages of CD34+ cell maturation, the distribution of CD164 is shifted toward lysosomal compartments, where it is unlikely to bind its ligand. The mechanisms whereby sorting CD164 to different endocytic compartments at different stages of maturation of hematopoietic precursor cells is accomplished are not clear. However, a mechanism might be operating by which the binding affinities of the YXXø motif in the cytoplasmic tail for different adaptor protein complexes can be regulated, e.g. by cytokine receptor interactions in cis. This would lead to changes in the surface and intracellular distribution of CD164. We have previously observed that the class II 103B2/9E10 epitope is more rapidly down-regulated from the cell surface than the class III N6B6 epitope (6, 8). Interestingly, a second small population of KG1B cells, presumably representing more mature cells, did not express the class II epitope at all or expressed it at very low levels. This suggests that another regulatory mechanism exists that influences the glycosylation pattern of CD164 and thus the binding characteristics regarding its ligand(s). Whether different intracellular trafficking routes are regulated by differential glycosylation of CD164 or vice versa remains to be determined. However, it is possible that both events are interconnected and contribute to the regulation of CD164 function.

In this study, we have demonstrated that human and murine CD164/endolyn are highly conserved with respect to their glycoprotein and gene structures and their chromosomal locations. Furthermore, we show that, in both species, the major isoform is encoded by all six CD164 exons and that this maintains functionally important motifs that regulate cell proliferation or subcellular distribution. Thus, this is the first step in our aim to define the nature, expression, and regulated expression of the natural cellular ligands and binding partners for CD164. The availability in a variety of species of the genomic structures and of a defined set of CD164 cDNAs and antibodies plus the identification of functionally important modules and motifs will permit the critical definition of CD164 functions and will aid in determining structure, function, and signaling relationships both in vitro and in vivo.


    ACKNOWLEDGEMENTS

We thank Professors P. Sissons and A. C. Minson for access to the Leica confocal microscope, Dr. A. J. Thomson for advice on the RNase protection assay, Dr. K. Clark for advice on sequencing, and Dr. M. J. Clague and Professor C. R. Hopkins for providing antibodies. We also thank Professor D. J. Weatherall and Dr. J. P. Luzio for support.


    FOOTNOTES

* This work was supported by the Leukaemia Research Fund, the Medical Research Council, an Overseas Research Student award, and the Wellcome Trust, United Kingdom.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF299340 (human genomic CD164), AF299341 (human CD164(E1-6) cDNA), AF299342 (human CD164(Delta E5)), AF299343 (human CD164(Delta E4)), AF299344 (mouse genomic CD164), and AF299345 (mouse CD164(E1-6)).

** To whom correspondence should be addressed: MRC Molecular Hematology, Inst. of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom. Tel.: 44-1865-222-632; Fax: 44-1865-222-500; E-mail: Swatt@molbiol.ox.ac.uk.

Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M007965200

2 C. R. Hopkins, unpublished data.

3 S. M. Watt, unpublished data.


    ABBREVIATIONS

The abbreviations used are: mAb, monoclonal antibody; GM-CSF, granulocyte/macrophage colony-stimulating factor; IL, interleukin; G-CSF, granulocyte colony-stimulating factor; mIg, murine Ig; PAC, P1 artificial chromosome; RT-PCR, reverse transcription-polymerase chain reaction; contig, group of overlapping clones; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UTR, untranslated region; 3'-RACE, 3'-rapid amplification of cDNA ends; kb, kilobase pair(s); bp, base pair(s); BFU-E, erythroid burst-forming unit(s); GM-CFC, granulocte/macrophage colony-forming cells.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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