Distinct Kinetic and Molecular Requirements Govern CD44 Binding to Hyaluronan versus Fibrin(ogen)

CD44 is a multifunctional glycoprotein that binds to hyaluronan and fibrin(ogen). Alternative splicing is responsible for the generation of numerous different isoforms, the smallest of which is CD44s. Insertion of variant exons into the extracellular membrane proximal region generates the variant isoforms (CD44v). Here, we used force spectroscopy to delineate the biophysical and molecular requirements of CD44-HA and CD44-fibrin(ogen) interactions at the single-molecule level. CD44v-HA and CD44s-HA single bonds exhibit similar kinetic and micromechanical properties because the HA-binding motif on CD44 is common to all of the isoforms. Although this is the primary binding site, O- and N-linked glycans and sulfation also contribute to the tensile strength of the CD44-HA bond. The CD44s-fibrin pair has a lower unstressed dissociation rate and a higher tensile strength than CD44s-fibrinogen but is weaker than the CD44-HA bond. In contrast to CD44-HA binding, the molecular interaction between CD44 and fibrin(ogen) is predominantly mediated by the chondroitin sulfate and dermatan sulfate on CD44. Blocking sulfation on CD44s modestly decreases the tensile strength of CD44s-fibrin(ogen) binding, which is in stark contrast to CD44v-fibrin interaction. Collectively, the results obtained by force spectroscopy in conjunction with biochemical interventions enable us to delineate the biophysical parameters and molecular constituents of CD44 binding to hyaluronan and fibrin(ogen).     

Expression of Recombinant Hyaluronan Synthase (HAS) Isoforms in CHO Cells Reduces Cell Migration and Cell Surface CD44

In the present study we investigated the functional properties of the three recombinant hyaluronan synthases (HAS proteins) HAS1, HAS2, and HAS3. HAS3-transfected CHO clones exhibited the highest hyaluronan polymerization rate followed by HAS2 transfectants which were more catalytically active than HAS1 transfectants. In living cells all three HAS proteins synthesized hyaluronan chains of high molecular weight (larger than 3.9 x 10(6)). In vitro, the HAS2 isoform produced hyaluronan chains of a molecular weight larger than 3.9 x 10(6), whereas HAS3 produced polydisperse hyaluronan (molecular weight 0.12-1 x 10(6)), and HAS1 synthesized much shorter chains of an average molecular weight of 0.12 x 10(6). Thus, each HAS protein may interact with different cytoplasmic proteins which may influence their catalytic activity. CHO transfectants with the ability to synthesize about 1 microgram hyaluronan/1 x 10 (5) cells/24 h were surrounded by hyaluronan-containing coats, whereas transfectants generating about 4-fold lower amounts of hyaluronan formed coats only in the presence of chondroitin sulfate proteoglycan. An inverse correlation between hyaluronan production on the one hand and cell migration and cell surface CD44 expression on the other was found; a 4-fold lower migration and a 2-fold decrease of cell surface CD44 receptors was seen when hyaluronan production increased 1000-fold over the level in the untransfected cells. The inverse relationships between hyaluronan production and migration and CD44 expression of cells are of importance for the regulation of cell-extracellular matrix interactions.     

Glycosylation of CD44 is implicated in CD44-mediated cell adhesion to hyaluronan

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.     

Discrete Domains Within the Hyaluronan Receptor CD44 Regulate Membrane Localization and Cell Migration

CD44 is the principle transmembrane receptor for the extracellular matrix glycosaminoglycan, hyaluronan. This receptor: ligand interaction is required for many normal cellular processes including lymphocyte homing into inflammatory sites, assembly of a pericellular matrix during chondrogenesis, wound healing and tissue morphogenesis during development. In order to mediate these diverse events, CD44 expressing cells must be able to regulate, and respond to, interactions with hyaluronan. The mechanisms responsible have been subject to scrutiny over the past few years as it has become clear that their disruption can underlie the progression of both metastatic tumours and chronic inflammatory diseases. Here we describe recent data identifying discrete regions within the transmembrane and cytoplasmic domains of CD44 which regulate this important adhesion receptor.     

Hyaluronan oligosaccharides induce CD44 cleavage and promote cell migration in CD44-expressing tumor cells

CD44 is an adhesion molecule that serves as a cell surface receptor for several extracellular matrix components, including hyaluronan (HA). The proteolytic cleavage of CD44 from the cell surface plays a critical role in the migration of tumor cells. Although this cleavage can be induced by certain stimuli such as phorbol ester and anti-CD44 antibodies in vitro, the physiological inducer of CD44 cleavage in vivo is unknown. Here, we demonstrate that HA oligosaccharides of a specific size range induce CD44 cleavage from tumor cells. Fragmented HA containing 6-mers to 14-mers enhanced CD44 cleavage dose-dependently by interacting with CD44, whereas a large polymer HA failed to enhance CD44 cleavage, although it bound to CD44. Examination using uniformly sized HA oligosaccharides revealed that HAs smaller than 36 kDa significantly enhanced CD44 cleavage. In particular, the 6.9-kDa HA (36-mers) not only enhanced CD44 cleavage but also promoted tumor cell motility, which was completely inhibited by an anti-CD44 monoclonal antibody. These results raise the possibility that small HA oligosaccharides, which are known to occur in various tumor tissues, promote tumor invasion by enhancing the tumor cell motility that may be driven by CD44 cleavage.     

The High and Low Molecular Weight Forms of Hyaluronan Have Distinct Effects on CD44 Clustering

CD44 is a major cell surface receptor for the glycosaminoglycan hyaluronan (HA). Native high molecular weight hyaluronan (nHA) and oligosaccharides of hyaluronan (oHA) provoke distinct biological effects upon binding to CD44. Despite the importance of such interactions, however, the feature of binding with CD44 at the cell surface and the molecular basis for functional distinction between different sizes of HA is still unclear. In this study we investigated the effects of high and low molecular weight hyaluronan on CD44 clustering. For the first time, we provided direct evidence for a strong relationship between HA size and CD44 clustering in vivo. In CD44-transfected COS-7 cells, we showed that exogenous nHA stimulated CD44 clustering, which was disrupted by oHA. Moreover, naturally expressed CD44 was distributed into clusters due to abundantly expressed nHA in HK-2 cells (human renal proximal tubule cells) and BT549 cells (human breast cancer cell line) without exogenous stimulation. Our results suggest that native HA binding to CD44 selectively induces CD44 clustering, which could be inhibited by oHA. Finally, we demonstrated that HA regulates cell adhesion in a manner specifically dependent on its size. oHA promoted cell adhesion while nHA showed no effects. Our results might elucidate a molecular- and/or cellular-based mechanism for the diverse biological activities of nHA and oHA.     

D1 Dopamine Receptors Can Interact with Both Stimulatory and Inhibitory Guanine Nucleotide Binding Proteins

Pretreatment of striatal membranes with N-ethylmaleimide in the presence of a D1-specific agonist inactivated endogenous guanine nucleotide binding proteins (G proteins), but not D1 dopamine receptors, resulting in a loss of high-affinity agonist binding sites. Such D1 receptors were solubilized, mixed with exogenous G proteins from cells not containing D1 receptors, and reconstituted into phospholipid vesicles. These reconstituted receptors were able to couple to the exogenous G proteins, and the proportion of agonist high-affinity sites of the receptor (40-57%) was similar to levels obtained with naive receptors coupling to endogenous G proteins (40%) upon solubilization and reconstitution. These hybrid high-affinity sites were fully modulated by guanine nucleotides. Pretreatment of cells with pertussis toxin prior to extraction of G proteins resulted in a 50% decrease in the proportion of high-affinity sites; these sites remained sensitive to guanine nucleotides. When D1 receptors were reconstituted with extracts of cyc- cells, which lack stimulatory G proteins, the proportion of high-affinity sites was reduced to 31% of the total. Pertussis toxin treatment of the cyc- cells completely abolished the formation of high-affinity sites. These results demonstrate that D1-dopaminergic receptors are able to couple to not only stimulatory G proteins (Gs), but also to inhibitory G proteins (Gi).     

Induction of a Hyaluronan Receptor,CD44, during Embryonal Carcinoma and Embryonic Stem Cell Differentiation

This paper describes the expression profile of the CD44 glycoprotein during differentiation of embryonal carcinoma (EC) and embryonic stem (ES) cells. We have recently shown that CD44 is expressed in discrete embryonic structures and, in view of this, we sought an in vitro differentiation model of development in which we could study more readily the structure and function of the CD44 molecule. The P19 EC and CGR8 ES cells were chosen as they have the capacity to develop down the cardiac muscle pathway and we have previously demonstrated that CD44 is expressed abundantly in the embryonic myocardium. The differentiation process in both cell types is accompanied by an induction of CD44 mRNA and protein. However, in differentiated cultures CD44 is not expressed in contractile cells, indicating that these P19 cells do not represent CD44-positive embryonic cardiomyocytes. Expression of CD44 is observed on fibroblast-like cells which appear to migrate over and out from the plated aggregates. Hyaluronan, the major ligand for CD44, is also associated with these CD44-positive fibroblast-like cells. It is suggested that expression of both receptor and ligand by the fibroblastic cells is required for cell:matrix adhesion and cell motility. As CD44 is up-regulated in these cultures, P19 cells are now established as a useful model system to study the factors regulating expression of the CD44 gene.     

Ras-Transformed Cells Express Both CD44 and RHAMM Hyaluronan Receptors: Only RHAMM Is Essential for Hyaluronan-Promoted Locomotion

Hyaluronan (HA) is an important regulator of cell locomotion. We show that ras -transformed cells, termed 245 cells, respond to HA with an increase in random locomotion. We show that two HA receptors, RHAMM (receptor for hyaluronan-mediated motility) and CD44, are present on these ras -transformed fibroblasts. RHAMM is expressed as a 58-kDa protein and is distributed primarily as patches over lamellae. CD44 occurs largely as an 85- to 90-kDa protein that is distributed more or less evenly over the cell surface with small amounts concentrated at the tips of lamellae. CD44 and RHAMM both bind biotinylated HA in a transblot assay, indicating that they are both potential fibroblast HA receptors. CD44 binds approximately five times more HA than RHAMM as determined by densitometric analysis of transblots, indicating that this protein is the major HA receptor on fibroblasts. We assessed the role of these receptors in mediating the stimulatory effects of HA on cell motility by using antibody neutralization. Several antibodies to CD44 were used that inhibit HA/CD44 interactions. None of these had an effect on locomotory responses to HA, indicating that CD44 is not directly involved in mediating locomotion in response to HA on ras-transformed cells. In contrast, antibodies specific to RHAMM completely inhibited locomotion, indicating that RHAMM is the primary mediator of HA-promoted locomotion of ras -transformed cells.     

Involvement of Opioid Receptor Subtypes in Both Stimulatory and Inhibitory Effects of the Opioid Peptides on Prolactin Secretion During Pregnancy

1. We have previously demonstrated the existence of a dual neuromodulatory regulation of prolactin secretion by the opioid system. In the present work, we evaluated the opioid receptor subtypes involved in both the stimulatory and the inhibitory regulation of prolactin secretion in pregnant rats. 2. Specific opioid agonists and antagonists were administered intracerebro ventricular (i.c.v.) to rats on day 3 and on day 19 pregnancy in rats of pretreated with mifepristone. Blood samples were obtained after decapitation at 12.00 and 18.00 h. Serum prolactin levels were measured by RIA. 3. The mu-selective agonist DAMGO and beta-endorphin caused a significant increase in serum prolactin secretion on day 3 of pregnancy, during the diurnal surge and intersurge period. Pretreatment with naloxone prevented the increase on prolactin levels induced by DAMGO. The administration of U-50,488, a kappa-selective agonist or DPDPE, a delta-selective agonist, did not modify serum prolactin concentration while the mu1-antagonist naloxonazine reduced significantly serum prolactin levels. On day 19 of pregnancy, the release of prolactin induced by mifepristone was significantly increase by naloxonazine, while the kappa-antagonist nor-binaltorfimine induced only a small but significant increase. No effect was observed after administration of the delta-antagonist naltrindole. 4. We conclude that the mu-opioid receptor seems to be more specifically involved in both the stimulatory and inhibitory regulation by the opioid system on prolactin secretion during pregnancy. The increase on serum prolactin levels on day 3 after administration of DAMGO and beta-endorphin may suggest the participation of other regulatory mechanisms as the dopaminergic and serotoninergic systems. On day 19, only the endogenous ligands delta did not participate in the regulation of prolactin secretion, while the participation of the kappa-opioid receptor was significantly less effective than the endogenous ligand mu. Our results provide evidences of an important role of the opioid system through specific receptors on the regulation of prolactin secretion during early and late pregnancy.     

Hyaluronan and CD44 antagonize mitogen-dependent cyclin D1 expression in mesenchymal cells

High molecular weight (HMW) hyaluronan (HA) is widely distributed in the extracellular matrix, but its biological activities remain incompletely understood. We previously reported that HMW-HA binding to CD44 antagonizes mitogen-induced S-phase entry in vascular smooth muscle cells (SMCs; Cuff, C.A., D. Kothapalli, I. Azonobi, S. Chun, Y. Zhang, R. Belkin, C. Yeh, A. Secreto, R.K. Assoian, D.J. Rader, and E. Puré. 2001. J. Clin. Invest. 108:1031-1040); we now characterize the underlying molecular mechanism and document its relevance in vivo. HMW-HA inhibits the mitogen-dependent induction of cyclin D1 and down-regulation of p27(kip1) in vascular SMCs. p27(kip1) messenger RNA levels were unaffected by HMW-HA, but the expression of Skp2, the rate-limiting component of the SCF complex that degrades p27(kip1), was reduced. Rescue experiments identified cyclin D1 as the primary target of HMW-HA. Similar results were observed in fibroblasts, and these antimitogenic effects were not detected in CD44-null cells. Analysis of arteries from wild-type and CD44-null mice showed that the effects of HMW-HA/CD44 on cyclin D1 and Skp2 gene expression are detected in vivo and are associated with altered SMC proliferation after vascular injury.     

胃泌素C-端四肽的N-乙酰化和糖化及其对泌酸活性的影响

本文报道了胃泌素C—端四肽N—乙酰化和糖化的方法,并用大白鼠胃灌流技术研究了它们对泌酸活性的影响。结果表明,乙酰化和糖化均可提高其泌酸活性,其中葡萄糖—1—脱氧果糖四肽(Glc—1—deoxyfru—Trp—Met—Asp—Phe—NH_2)的泌酸活性超过了商品五肽胃泌素(Boc—α—Ala—Trp—Met—Asp—Phe—NH_2)     

Screening of Peptides Bound to Breast Cancer Stem Cell Specific Surface Marker CD44 by Phage Display

CD44, a cancer-associated membrane glycoprotein involved in cell adhesion and tumor progression, has been implicated as a cancer stem cell antigen in several cancers including breast cancer. If the detection sensitivity of CD44 as an early marker for cancer could be improved, this would have important clinical applications. As compared with early stage treatments of other kinds of cancer, treatment of breast cancer is more likely to results in positive outcomes, so this early detection is crucial. Therefore, CD44 is a potential diagnostic target for cancer detection. Herein, we have used a peptide library to screen novel diverse peptides that bind to CD44 with high affinity and characterized the specific binding of these peptides. Our work provides a basis to develop novel diagnostic peptides which may replace antibodies as CD44 detection probes.     

Hyaluronan recognition mode of CD44 revealed by cross-saturation and chemical shift perturbation experiments

CD44 is the main cell surface receptor for hyaluronic acid (HA) and contains a functional HA-binding domain (HABD) composed of a Link module with N- and C-terminal extensions. The contact residues of human CD44 HABD for HA have been determined by cross-saturation experiments and mapped on the topology of CD44 HABD, which we elucidated by NMR. The contact residues are distributed in both the consensus fold for the Link module superfamily and the additional structural elements consisting of the flanking regions. Interestingly, the contact residues exhibit small changes in chemical shift upon HA binding. In contrast, the residues with large chemical shift changes are localized in the C-terminal extension and the first alpha-helix and are generally inconsistent with the contact residues. These results suggest that, upon ligand binding, the C-terminal extension and the first alpha-helix undergo significant conformational changes, which may account for the broad ligand specificity of CD44 HABD.     

Inhibitory Effects of a Pectin-Enriched Tomato Cell Wall Fraction on Agrobacterium tumefaciens Binding and Tumor Formation

A pectin-enriched soluble cell wall fraction (CWF) prepared from suspension cultured tomato cells inhibits binding of Agrobacterium tumefaciens to these cells. It was hypothesized that the CWF contains the plant surface binding site for A. tumefaciens (NT Neff, AN Binns 1985 Plant Physiol 77: 35-42). Experiments described here demonstrate that tomato CWF inhibited tumor formation on potato slices and Agrobacterium binding to intact tomato cells in a dose-dependent fashion. Boiling the fraction reduced both its binding and tumor inhibitory activities. Tumor inhibitory activity was titrated out by increased concentrations of bacterial inocula with no inhibition apparent at 1 × 10⁸ bacteria per milliliter. These results indicate that a tomato CWF is enriched for a putative A. tumefaciens binding site which may also be involved in tumor formation in potato.     

Binding of Sindbis Virus to Cell Surface Heparan Sulfate

Alphaviruses are arthropod-borne viruses with wide species ranges and diverse tissue tropisms. The cell surface receptors which allow infection of so many different species and cell types are still incompletely characterized. We show here that the widely expressed glycosaminoglycan heparan sulfate can participate in the binding of Sindbis virus to cells. Enzymatic removal of heparan sulfate or the use of heparan sulfate-deficient cells led to a large reduction in virus binding. Sindbis virus bound to immobilized heparin, and this interaction was blocked by neutralizing antibodies against the viral E2 glycoprotein. Further experiments showed that a high degree of sulfation was critical for the ability of heparin to bind Sindbis virus. However, Sindbis virus was still able to infect and replicate on cells which were completely deficient in heparan sulfate, indicating that additional receptors must be involved. Cell surface binding of another alphavirus, Ross River virus, was found to be independent of heparan sulfate.     

Hyaluronan and CD44: strategic players for cell-matrix interactions during chondrogenesis and matrix assembly

Embryonic induction, soluble and insoluble factors, receptors, and signal transduction are orchestrated for the morphogenesis of the cartilage elements. The interaction of cells with the extracellular matrix (ECM) may lead to altered cellular response to morphogens based on the formation of new adhesive contacts, or the uncoupling of cell-matrix interactions. Hyaluronan's influence on cell behavior, and its intimate association with cells are accomplished by a wide variety of specific binding proteins for hyaluronan. The temporal expression of the hyaluronan receptor CD44 (which is expressed as several alternatively spliced variants) may be strategic to many of these cell-matrix interactions during chondrogenesis. CD44 expression is temporally coincident with the reduction of intercellular spaces at the regions of future cartilage deposition. The spatial organization of CD44 at the cell surface may function to establish or regulate the structure of the pericellular matrix dependent on a hyaluronan scaffold. As the ECM is modified during embryogenesis, the cellular response to inductive signals may be altered. An uncoupling of chondrocyte-hyaluronan interaction leads to chondrocytic chondrolysis. Thus, consideration of cell-matrix interactions during chondrogenesis, in the light of our current understanding of the temporal and spatial expression of signaling morphogens, should become a promising focus of future research endeavors.     

PDGF Suppresses the Sulfation of CD44v and Potentiates CD44v-Mediated Binding of Colon Carcinoma Cells to Fibrin under Flow

Fibrin(ogen) mediates sustained tumor cell adhesion and survival in the pulmonary vasculature, thereby facilitating the metastatic dissemination of tumor cells. CD44 is the major functional fibrin receptor on colon carcinoma cells. Growth factors, such as platelet-derived growth factor (PDGF), induce post-translational protein modifications, which modulate ligand binding activity. In view of the roles of PDGF, fibrin(ogen) and CD44 in cancer metastasis, we aimed to delineate the effect of PDGF on CD44-fibrin recognition. By immunoprecipitating CD44 from PDGF-treated and untreated LS174T colon carcinoma cells, which express primarily CD44v, we demonstrate that PDGF enhances the adhesion of CD44v-coated beads to immobilized fibrin. Enzymatic inhibition studies coupled with flow-based adhesion assays and autoradiography reveal that PDGF augments the binding of CD44v to fibrin by significantly attenuating the extent of CD44 sulfation primarily on chondroitin and dermatan sulfate chains. Surface plasmon resonance assays confirm that PDGF enhances the affinity of CD44v-fibrin binding by markedly reducing its dissociation rate while modestly increasing the association rate. PDGF mildly reduces the affinity of CD44v-hyaluronan binding without affecting selectin-CD44v recognition. The latter is attributed to the fact that CD44v binds to selectins via sialofucosylated O-linked residues independent of heparan, dermatan and chondroitin sulfates. Interestingly, PDGF moderately reduces the sulfation of CD44s and CD44s-fibrin recognition. Collectively, these data offer a novel perspective into the mechanism by which PGDF regulates CD44-dependent binding of metastatic colon carcinoma cells to fibrin(ogen).