The expression of human blood group antigens during erythropoiesis in a cell culture system.
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Phenotypic analysis of hematopoietic stem and progenitor cells has been an invaluable tool in defining the biology of stem cell populations. We use here flow cytometry to examine the expression of human erythroid-specific surface markers during the maturation of early committed erythroid cells derived from cord blood in vitro. The temporal order of the expression of erythroid specific markers was as follows: Kell glycoprotein (gp), Rh gp, Landsteiner Wiener (LW) gp, glycophorin A (GPA), Band 3, Lutheran (Lu) gp, and Duffy (Fy) gp. The time at which some of these markers appeared suggests possible roles for some of these erythroid-specific polypeptides during the differentiation of these committed progenitors. The early appearance of Kell gp raises the possibility that it may have an important role in the early stages of hematopoiesis or cell lineage determination. Kell gp may also be a useful marker for the diagnosis of erythroleukemia. The late expression of Lu gp suggests it may be involved in the migration of erythroid precursors from the marrow. Fy gp is also expressed late consistent with a role as a scavenger receptor for cytokines in the bone marrow and circulation. Rh c antigen appeared before Rh D antigen, and it is suggested that this may reflect a reorganization of the developing erythroid cell membrane involving the Rh polypeptides and other components, including GPA and Band 3. (+info)
Isoforms of the Lutheran/basal cell adhesion molecule glycoprotein are differentially delivered in polarized epithelial cells. Mapping of the basolateral sorting signal to a cytoplasmic di-leucine motif.
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Lu and Lu(v13) are two glycoprotein (gp) isoforms that belong to the immunoglobulin superfamily and carry both the Lutheran (Lu) blood group antigens and the basal cell adhesion molecule epithelial cancer antigen. Lu (85 kDa) and Lu(v13) (78 kDa) gps, which differ only in the length of their cytoplasmic domain, are adhesion molecules that bind laminin. In nonerythroid tissues, the Lu/basal cell adhesion molecule antigens are predominantly expressed in the endothelium of blood vessel walls and in the basement membrane region of normal epithelial cells, whereas they exhibit a nonpolarized expression in some epithelial cancers. Here, we analyzed the polarization of Lu and Lu(v13) gps in epithelial cells by confocal microscopy and domain-selective biotinylation assays. Differentiated human colon carcinoma Caco-2 cells exhibited a polarized expression of endogenous Lu antigens associated with a predominant expression of the Lu isoform at the basolateral domain of the plasma membrane and a very low expression of the Lu(v13) isoform at both the apical and basolateral domains. Analysis of transfected Madin-Darby canine kidney cells revealed a basolateral expression of Lu gp and a nonpolarized expression of Lu(v13) gp. Delivery of Lu(v13) to both apical and basolateral surfaces showed similar kinetics, indicating that this isoform is directly transported to each surface domain. A dileucine motif at position 608-609, specific to the Lu isoform, was characterized as a dominant basolateral sorting signal that prevents Lu gp from taking the apical delivery pathway. (+info)
Selective Lutheran glycoprotein gene expression at the blood-brain barrier in normal brain and in human brain tumors.
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The Lutheran (LU) glycoprotein was shown to be a specific marker of brain capillary endothelium, which forms the blood-brain barrier (BBB) in vivo. A 1.5 kb partial cDNA encoding the bovine LU was isolated from a bovine brain capillary cDNA library. Sequence analysis showed that the bovine and human LU had a 75% and 79% identity in the amino acid and nucleotide sequences, respectively. Northern blot analysis demonstrated a very high level of gene expression of the LU transcript in freshly isolated bovine brain capillaries, but no measurable LU mRNA in whole bovine brain. The high level of LU gene expression was maintained when bovine brain capillary endothelium was grown in tissue culture. Because many BBB specific genes are downregulated in tissue culture and in brain tumors, the expression of the LU mRNA and immunoactive LU protein was investigated in primary and metastatic human brain tumors. Immunocytochemistry of fresh frozen human brain and human brain tumors showed abundant immunostaining of brain capillary endothelium. Northern blot analysis showed the presence of LU transcripts in a panel of primary and metastatic human brain tumors. These studies demonstrated that the LU glycoprotein was a novel new marker of the BBB, and unlike other BBB specific genes, there was a persistent gene expression of the LU glycoprotein both in brain capillary endothelial cells grown in culture and in the endothelium of capillaries perfusing human brain cancer. (+info)
Basal-cell adhesion molecule (B-CAM) is induced in epithelial skin tumors and inflammatory epidermis, and is expressed at cell-cell and cell-substrate contact sites.
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Basal-cell adhesion molecule (B-CAM) is a 90 kDa cell surface glycoprotein of the immunoglobulin superfamily that functions as a laminin-binding receptor. B-CAM is upregulated following malignant transformation of some cell types in vivo and in vitro, thus being a candidate molecule involved in tumor progression. As cutaneous distribution and function of B-CAM are largely unknown, we have studied its expression and regulation in normal and diseased human skin. In normal skin, B-CAM was expressed by endothelial cells of dermal blood vessels. In contrast, B-CAM was strongly upregulated within the tumor tissue of both malignant and benign epithelial skin tumors, including basal cell carcinomas, squamous cell carcinomas, keratoacanthomas, and common warts. Transformation-associated upregulation was confirmed in vitro, but normal keratinocytes also expressed B-CAM under culture conditions. Interestingly, the basal epidermal layer of normal-appearing skin surrounding the tumors also expressed B-CAM, and B-CAM were induced on the basal and apicolateral surfaces of basal keratinocytes in inflammatory skin disorders suggesting transformation-independent mechanisms of epidermal induction of the B-CAM. Immunoelectron microscopy studies of cultured transformed keratinocytes revealed that B-CAM was expressed at cell-cell and cell-substrate contact sites. Halting proliferation of transformed keratinocytes through cytostatic drugs resulted in decreased B-CAM synthesis. Likewise, inducing terminal differentiation in keratinocyte cultures by increasing the Ca(2+) concentration in the medium decreased B-CAM expression. In contrast, both ultraviolet A and B irradiation of cultured human keratinocytes resulted in significantly increased expression of the B-CAM. Overall, it appears that B-CAM expression in human skin is associated with activated states of keratinocytes, and that B-CAM may be involved in cell-cell adhesion or migration, in addition to its known function as a laminin receptor. J Invest Dermatol 115:1047-1053 2000 (+info)
Lutheran blood group glycoprotein and its newly characterized mouse homologue specifically bind alpha5 chain-containing human laminin with high affinity.
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Lutheran blood group glycoproteins (Lu gps) are receptors for the extracellular matrix protein, laminin. Studies suggest that Lu gps may contribute to vaso-occlusion in sickle cell disease and it has recently been shown that sickle cells adhere to laminin isoforms containing the alpha5 chain (laminin 10/11). Laminin alpha5 is present in the subendothelium and is also a constituent of bone marrow sinusoids, suggesting a role for the Lu/laminin interaction in erythropoiesis. The objectives of the current study were to define more precisely the molecular interactions of the extracellular and intracellular regions of human Lu and to clone and characterize a mouse homologue. To this end, complementary DNA and genomic clones for the mouse homologue were sequenced and the mouse Lu gene mapped to a region on chromosome 7 with conserved synteny with human 19q13.2. Mouse and human Lu gps are highly conserved (72% identity) at the amino acid sequence level and both mouse and human Lu gps specifically bind laminin 10/11 with high affinity. Furthermore, the first 3, N-terminal, immunoglobulin superfamily domains of human Lu are critical for this interaction. The results indicated that the cytoplasmic domain of BRIC 221-labeled human Lu gp is linked with the spectrin-based skeleton, affording the speculation that this interaction may be critical for signal transduction. These results further support a role for Lu gps in sickle cell disease and indicate the utility of mouse models to explore the function of Lu gp-laminin 10/11 interaction in normal erythropoiesis and in sickle cell disease. (+info)
Characterization of the laminin binding domains of the Lutheran blood group glycoprotein.
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Lutheran (Lu) blood group antigens and the basal cell adhesion molecule antigen reside on two glycoproteins that belong to the Ig superfamily (IgSF) and carry five Ig-like extracellular domains. These glycoproteins act as widely expressed adhesion molecules and represent the unique receptors for laminin-10/11 in erythroid cells. Here, we report the mapping of IgSF domains responsible for binding to laminin. In plasmonic resonance surface experiments, only recombinant Lu proteins containing the N-terminal IgSF domains 1-3 were able to bind laminin-10/11 and to inhibit binding of laminin to Lu-expressing K562 cells. Mutant recombinant proteins containing only IgSF domain 1, domains 1 + 2, domains 1 + 3, domains 2 + 3, domain 3, domain 4, domain 5, and domains 4 + 5 failed to bind laminin as well as a construct containing all of the extracellular domains except domain 3. Altogether, these results indicate that IgSF domains 1-3 are involved in laminin binding and that a specific spatial arrangement of these three first domains is most probably necessary for interaction. Neither the RGD nor the N-glycosylation motifs present in IgSF domain 3 were involved in laminin binding. (+info)
Association of the tetraspanin CD151 with the laminin-binding integrins alpha3beta1, alpha6beta1, alpha6beta4 and alpha7beta1 in cells in culture and in vivo.
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CD151 is a cell surface protein that belongs to the tetraspanin superfamily. It forms complexes with the laminin-binding integrins alpha3beta1, alpha6beta1 and alpha6beta4 and is codistributed with these integrins in many tissues at sites of cell-matrix interactions. In this study we show that CD151 can also form stable complexes with the laminin-binding integrin alpha7beta1. The strength of this interaction is comparable to that between CD151 and alpha3beta1. Complexes of alpha3beta1, alpha6beta1 and alpha7beta1 with CD151 are equally well formed with all splice variants of the alpha3, alpha6 and alpha7 subunits, and complex formation is not affected by mutations that prevent the cleavage of the integrin alpha6 subunit. Like the expression of alpha3beta1 and alpha6beta1, expression of alpha7beta1 in K562 cells results in increased levels of CD151 at its surface. Two non-integrin laminin receptors, dystroglycan and the polypeptide on which the Lutheran blood group antigens are expressed, are also often colocalized with CD151, but no association with CD151-alpha3beta1 complexes was found with biochemical analysis. The anti-CD151 antibody TS151R detects an epitope at a site at which CD151 interacts with integrins, and therefore it cannot react with CD151 when it is bound to an integrin. Comparison of the straining patterns produced by TS151R with that by of an anti-CD151 antibody recognizing an epitope outside the binding site (P48) revealed that most tissues expressing one or more laminin-binding integrins reacted with P48 but not with TS151R. However, smooth muscle cells that express alpha7beta1 and renal tubular epithelial cells that express alpha6beta1 were stained equally well by TS151R and P48. These results suggest that the interactions between CD151 and laminin-binding integrins are subject to cell-type-specific regulation. (+info)
Vascular proteomics and subtractive antibody expression cloning.
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The cloning of genes expressing proteins that are differentially expressed in the organ microvasculature has the potential to address a variety of problems ranging from the analysis of disease pathogenesis to drug targeting for particular tissues. This study describes a methodology designed to analyze differential protein expression in the brain microvasculature. The method can be applied to other organs and is particularly suited to the cloning of cDNAs encoding membrane proteins. The technology merges a tissue-specific polyclonal antiserum with a cDNA library expression cloning system. The tissue-specific antiserum is subtracted with protein extracts from control tissues to remove those antibodies that recognize common antigenic proteins. Then, the depleted antiserum is used to expression clone tissue-specific proteins from a cDNA library expressed in mammalian cells. The methodology was evaluated with a rabbit polyclonal antiserum prepared against purified bovine brain capillaries. The antiserum was absorbed with acetone powders of liver and kidney and then used to screen a bovine brain capillary cDNA library in COS cells. The initial clone detected with this expression methodology was the Lutheran membrane glycoprotein, which is specifically expressed at the brain microvasculature compared with liver and kidney tissues. This subtractive expression cloning methodology provides a new approach to "vascular proteomics" and to the detection of proteins specifically expressed at the microvasculature, including membrane proteins. (+info)