Abstract. CD44-mediated cell adhesion to hyaluronate is controlled by mechanisms which are poorly understood. In the present work we examine the role of N-linked glycosylation and Ser-Gly motifs in regulating CD44-hyaluronate interaction. Our results show that treatment of a panel of human cell lines which constitutively express CD44 with the inhibitor of N-linked glycosylation tunicamycin results in the loss of attachment of these cells to hyaluronate-coated substrate. In contrast, treatment of the same cells with deoxymannojirimycin, which inhibits the conversion of high mannose oligosaccharides to complex N-linked carbohydrates, results in either no change or an increase in CD44-mediated adhesion to hyaluronate, suggesting that complex N-linked oligosaccharides may not be required for and may even inhibit CD44-HA interaction. Using human melanoma cells stably transfected with CD44 N-linked glycosylation site-specific mutants, we show that integrity of five potential N-linked glycosylation sites within the hyaluronate recognition domain of CD44 is critical for hyaluronate binding. Mutation of any one of these potential N-linked glycosylation sites abrogates CD44-mediated melanoma cell attachment to hyaluronate-coated surfaces, suggesting that all five sites are necessary to maintain the HA-recognition domain in the appropriate conformation. We also demonstrate that mutation of serine residues which constitute the four Ser-Gly motifs in the membrane proximal domain, and provide potential sites for glycosaminoglycan side chain attachment, impairs hyaluronate binding. Taken together, these observations indicate that changes in glycosylation of CD44 can have profound effects on its interaction with hyaluronic acid and suggest that glycosylation may provide an important regulatory mechanism of CD44 function.

Erythroid progenitors differentiate in erythroblastic islands, bone marrow niches composed of erythroblasts surrounding a central macrophage. Evidence suggests that within islands adhesive interactions regulate erythropoiesis and apoptosis. We are exploring whether erythroid intercellular adhesion molecule 4 (ICAM-4), an immunoglobulin superfamily member, participates in island formation. Earlier, we identified ␣ V integrins as ICAM-4 counterreceptors. Because macrophages express ␣ V , ICAM-4 potentially mediates island attachments. To test this, we generated ICAM-4 knock-out mice and developed quantitative, live cell techniques for harvesting intact islands and for re-forming islands in vitro. We observed a 47% decrease in islands reconstituted from ICAM-4 null marrow compared to wild-type marrow. We also found a striking decrease in islands formed in vivo in knock-out mice. Further, peptides that block ICAM-4/␣ V adhesion produced a 53% to 57% decrease in reconstituted islands, strongly suggesting that ICAM-4 binding to macrophage ␣ V functions in island integrity. Importantly, we documented that ␣ V integrin is expressed in macrophages isolated from erythroblastic islands. Collectively, these data provide convincing evidence that ICAM-4 is critical in erythroblastic island formation via ICAM-4/␣ V adhesion and also demonstrate that the novel experimental strategies we developed will be valuable in exploring molecular mechanisms of erythroblastic island formation and their functional role in regulating erythropoiesis. IntroductionErythroid progenitors proliferate, differentiate, and enucleate within specialized bone marrow niches, termed erythroblastic islands. [1][2][3][4] These structural units are composed of developing erythroblasts surrounding a central macrophage. It is apparent from ultrastructural studies that extensive cell-cell interactions, both erythroblastmacrophage, as well as erythroblast-erythroblast, occur within these 3-dimensional structures. However, little is known regarding either the molecular nature or functional role of the specific adhesive interactions. We are exploring the potential function of erythroid ICAM-4, a recently characterized member of the immunoglobulin superfamily, in erythroblastic island formation. ICAM-4 expression is limited to erythroid and placental tissue 5 but, to date, there is no information on its role in erythropoiesis. We earlier identified ␣ 4 ␤ 1 and ␣ V family integrins as ICAM-4-binding partners. 6 Because macrophages express ␣ V and erythroblasts exhibit ␣ 4 ␤ 1 , ICAM-4 is an attractive candidate for mediating erythroblast-erythroblast interactions via ICAM-4/␣ 4 ␤ 1 binding and regulating adhesion of erythroblasts to central macrophages via ICAM-4/␣ V binding.ICAM-4, which carries the Lansteiner Wiener (LW) blood group antigen system, has strong sequence homology with other members of the ICAM protein superfamily. 7,8 It is composed of 2 extracellular immunoglobulin-like domains, an N-terminal I set and a membrane proximal I2 set, and a single membr...

Lutheran blood group glycoproteins (Lu gps) are receptors for the extracellular matrix protein, laminin. Studies suggest that Lu gps may contribute to vasoocclusion in sickle cell disease and it has recently been shown that sickle cells adhere to laminin isoforms containing the ␣5 chain (laminin 10/11). Laminin ␣5 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. IntroductionThe Lutheran blood group is composed of a complex set of antigens expressed on 2 integral membrane glycoprotein isoforms of 85 and 78 kd. 1,2 The complementary DNA (cDNA) encoding the 85-kd isoform has been cloned, 3 and the predicted structure is that of a type 1 membrane protein. There are 5 disulfide-bonded, extracellular, immunoglobulin superfamily (IgSF) domains, a single hydrophobic membrane span, and a cytoplasmic tail of 59 residues. 3 The composition of the extracellular IgSF domains puts Lutheran blood group glycoproteins (Lu gps) in the subset of adhesion molecules that includes the human tumor marker MUC18/ MCAM4 and the chicken neural adhesion molecule Gicerin. [4][5][6] Chicken gicerin binds neurite outgrowth factor, a variant of the extracellular matrix (ECM) protein laminin 7,8 and, interestingly, recent studies suggest that Lu gp also functions as a laminin receptor. [9][10][11] The Lu gp cytoplasmic tail contains an SH3 binding motif and 5 potential phosphorylation sites, consistent with receptor signaling function. Of note, differences in the structure of the cytoplasmic tail distinguish the 2 isoforms. The 78-kd isoform (also termed B-CAM 12 or Lu[v13] 13 ) is generated by alternative splicing of intron 13 and differs from the larger form by having a truncated cytoplasmic tail lacking the SH3 binding motif as well as the potential phosphorylation sites. A recent study in epithelial cells 14 suggests that the cytop...