Antibody‐induced activation of the hyaluronan receptor function of CD44 requires multivalent binding by antibody

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CD44 can function as an adhesion receptor that mediates leukocyte rolling on hyaluronan (HA). To study the contributions of different domains of the standard isoform of CD44 to cell rolling, a CD44-negative mouse T lymphoma AKR1 was transfected with wild type (WT) or mutated cDNA constructs. A parallel flow chamber was used to study the rolling behavior of CD44 transfectants on immobilized HA. For CD44WT transfectants, the fraction of cells that rolled and the rolling velocity was inversely proportional to the amount of cell surface CD44. When the cytoplasmic domain distal to Gly 305 or sequences that serve as binding sites for cytoskeletal linker proteins, were deleted or replaced with foreign sequences, no significant changes in the rolling behavior of mutant cells, compared with the transfectant expressing CD44WT, were observed. Transfectants lacking 64 amino acids of the cytoplasmic tail distal to Cys 295 adhered to HA but showed enhanced rolling at low shear forces. When 83 amino acids from the "non-conserved" membrane-proximal region of the CD44 extracellular domain were deleted, cells adhered firmly to the HA substrate and did not roll at any fluid shear force tested. Unlike wild type cells that exhibited a nearly homogenous distribution of CD44 on a smooth cell surface, cells expressing the non-conserved region deletion mutant accumulated CD44 in membrane protrusions. Disruption of the actin cytoskeleton with cytochalasin B precluded the formation of membrane protrusions, however, treated cells still adhered firmly to HA and did not roll. We conclude that interaction between the cytoplasmic domain of CD44 and the cytoskeleton is not required for cell rolling on immobilized ligand. The strong effect of deletion of the non-conserved region of the extracellular domain argues for a critical role of this region in CD44-dependent rolling and adhesion interactions with HA under flow.

Section: Structure and Ha Binding Properties Of Wt And Mutant Cd44 Exsupporting

confidence: 90%

Role of the Extracellular and Cytoplasmic Domains of CD44 in the Rolling Interaction of Lymphoid Cells with Hyaluronan under Physiologic Flow

Gál¹,

Lesley²,

Ko³

et al. 2003

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“…CD44 clustering or dimerization seems required for HA binding in some cell types, particularly in lymphocytes, as demonstrated by, e.g. the effect of multivalent but not monovalent CD44 antibodies (56,57). CD44 clustering may also be required for ERM activation (41).…”

Section: Discussionmentioning

confidence: 97%

Two Novel Functions of Hyaluronidase-2 (Hyal2) Are Formation of the Glycocalyx and Control of CD44-ERM Interactions

Duterme

Mertens-Strijthagen

Tammi

et al. 2009

It has long been predicted that the members of the hyaluronidase enzyme family have important non-enzymatic functions. However, their nature remains a mystery. The metabolism of hyaluronan (HA), their major enzymatic substrate, is also enigmatic. To examine the function of Hyal2, a glycosylphosphatidylinositol-anchored hyaluronidase with intrinsically weak enzymatic activity, we have compared stably transfected rat fibroblastic BB16 cell lines with various levels of expression of Hyal2. These cell lines continue to express exclusively the standard form (CD44s) of the main HA receptor, CD44. Hyal2, CD44, and one of its main intracellular partners, ezrin-radixin-moesin (ERM), were found to co-immunoprecipitate. Functionally, Hyal2 overexpression was linked to loss of the glycocalyx, the HA-rich pericellular coat. This effect could be mimicked by exposure of BB16 cells either to Streptomyces hyaluronidase, to HA synthesis inhibitors, or to HA oligosaccharides. This led to shedding of CD44, separation of CD44 from ERM, reduction in baseline level of ERM activation, and markedly decreased cell motility (50% reduction in a wound healing assay). The effects of Hyal2 on the pericellular coat and on CD44-ERM interactions were inhibited by treatment with the Na ؉ /H ؉ exchanger-1 inhibitor ethyl-N-isopropylamiloride. We surmise that Hyal2, through direct interactions with CD44 and possibly some pericellular hyaluronidase activity requiring acidic foci, suppresses the formation or the stability of the glycocalyx, modulates ERMrelated cytoskeletal interactions, and diminishes cell motility. These effects may be relevant to the purported in vivo tumorsuppressive activity of Hyal2.

“…Hyaluronan from rooster comb (Sigma) was fluorescein-conjugated with fluoresceinamine (46). Cellsurface binding of fluorescein-conjugated HA (FL-HA) was determined by flow cytometry (47). HA oligosaccharides HA 4 , HA 6 , HA 8 , HA 10 , HA 12 , HA 14 , and HA 16 were made as described (48).…”

Section: Xj(3)/cd44mentioning

confidence: 99%

Hyaluronan Binding Properties of a CD44 Chimera Containing the Link Module of TSG-6

Lesley¹,

English²,

Gál³

et al. 2002

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CD44, a cell-surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, can mediate leukocyte rolling on hyaluronan substrates and has been implicated in leukocyte migration to sites of inflammation. CD44-mediated binding to hyaluronan is of low affinity, and effective cell/matrix interaction depends on multiple interactions with the multivalent ligand. We replaced the Link module of CD44 with the homologous region of TSG-6, a hyaluronan-binding protein secreted in response to inflammation whose Link module has a higher affinity for ligand. Monoclonal antibodies raised against the CD44/TSG-6 chimera recognized recombinant human TSG-6 and native mouse TSG-6 and blocked hyaluronan binding to these proteins. Cells expressing the CD44/TSG-6 molecule bound hyaluronan with higher avidity than cells expressing CD44. This resulted in changes in the hyaluronan binding properties characteristic of cells expressing CD44 such as requirements for threshold levels of receptor expression and for hyaluronan of high molecular mass. In parallel plate flow assays used to model leukocyte rolling, cells expressing CD44/TSG-6 failed to roll on hyaluronan. Instead, they stuck and remained "tethered" to the substrate under fluid flow. This result argues that the low affinity of CD44 for its ligand is important for rolling, an early phase of leukocyte extravasation from the blood.