Structure of the regulatory hyaluronan binding domain in the inflammatory leukocyte homing receptor CD44

Adhesive interactions involving CD44, the cell surface receptor for hyaluronan, underlie fundamental processes such as inflammatory leukocyte homing and tumor metastasis. Regulation of such events is critical and appears to be effected by changes in CD44 N-glycosylation that switch the receptor "on" or "off" under appropriate circumstances. How altered glycosylation influences binding of hyaluronan to the lectin-like Link module in CD44 is unclear, although evidence suggests additional flanking sequences peculiar to CD44 may be involved. Here we show using X-ray crystallography and NMR spectroscopy that these sequences form a lobular extension to the Link module, creating an enlarged HA binding domain and a formerly unidentified protein fold. Moreover, the disposition of key N-glycosylation sites reveals how specific sugar chains could alter both the affinity and avidity of CD44 HA binding. Our results provide the necessary structural framework for understanding the diverse functions of CD44 and developing novel therapeutic strategies

Binding of Hyaluronan to the Native Lymphatic Vessel Endothelial Receptor LYVE-1 Is Critically Dependent on Receptor Clustering and Hyaluronan Organization

The lymphatic endothelial receptor LYVE-1 has been implicated in both uptake of hyaluronan (HA) from tissue matrix and in facilitating transit of leukocytes and tumor cells through lymphatic vessels based largely on in vitro studies with recombinant receptor in transfected fibroblasts. Curiously, however, LYVE-1 in lymphatic endothelium displays little if any binding to HA in vitro, and this has led to the conclusion that the native receptor is functionally silenced, a feature that is difficult to reconcile with its proposed in vivo functions. Nonetheless, as we reported recently, LYVE-1 can function as a receptor for HA-encapsulated Group A streptococci and mediate lymphatic dissemination in mice. Here we resolve these paradoxical findings and show that the capacity of LYVE-1 to bind HA is strictly dependent on avidity, demanding appropriate receptor self-association and/or HA multimerization. In particular, we demonstrate the prerequisite of a critical LYVE-1 threshold density and show that HA binding may be elicited in lymphatic endothelium by surface clustering with divalent LYVE-1 mAbs. In addition, we show that cross-linking of biotinylated HA in streptavidin multimers or supramolecular complexes with the inflammation-induced protein TSG-6 enables binding even in the absence of LYVE-1 cross-linking. Finally, we show that endogenous HA on the surface of macrophages can engage LYVE-1, facilitating their adhesion and transit across lymphatic endothelium. These results reveal LYVE-1 as a low affinity receptor tuned to discriminate between different HA configurations through avidity and establish a new mechanistic basis for the functions ascribed to LYVE-1 in matrix HA binding and leukocyte trafficking in vivo

Micromechanical Analysis of the Hyaluronan-Rich Matrix Surrounding the Oocyte Reveals a Uniquely Soft and Elastic Composition

The cumulus cell-oocyte complex (COC) matrix is an extended coat that forms around the oocyte a few hours before ovulation and plays vital roles in oocyte biology. Here, we analyzed the micromechanical response of mouse COC matrix by colloidal-probe atomic force microscopy. We found that the COC matrix is elastic insofar as it does not flow and its original shape is restored after force release. At the same time, the COC matrix is extremely soft. Specifically, the most compliant parts of in vivo and in vitro expanded COC matrices yielded Young's modulus values of 0.5 ± 0.1 Pa and 1.6 ± 0.3 Pa, respectively, suggesting both high porosity and a large mesh size (≥100 nm). In addition, the elastic modulus increased progressively with indentation. Furthermore, using optical microscopy to correlate these mechanical properties with ultrastructure, we discovered that the COC is surrounded by a thick matrix shell that is essentially devoid of cumulus cells and is enhanced upon COC expansion in vivo. We propose that the pronounced nonlinear elastic behavior of the COC matrix is a consequence of structural heterogeneity and serves important functions in biological processes such as oocyte transport in the oviduct and sperm penetration

Rapid Lymphatic Dissemination of Encapsulated Group A Streptococci <i>via</i> Lymphatic Vessel Endothelial Receptor-1 Interaction

<div><p>The host lymphatic network represents an important conduit for pathogen dissemination. Indeed, the lethal human pathogen group A streptococcus has a predilection to induce pathology in the lymphatic system and draining lymph nodes, however the underlying basis and subsequent consequences for disease outcome are currently unknown. Here we report that the hyaluronan capsule of group A streptococci is a crucial virulence determinant for lymphatic tropism <i>in vivo</i>, and further, we identify the lymphatic vessel endothelial receptor-1 as the critical host receptor for capsular hyaluronan in the lymphatic system. Interference with this interaction <i>in vivo</i> impeded bacterial dissemination to local draining lymph nodes and, in the case of a hyper-encapsulated M18 strain, redirected streptococcal entry into the blood circulation, suggesting a pivotal role in the manifestation of streptococcal infections. Our results reveal a novel function for bacterial capsular polysaccharide in directing lymphatic tropism, with potential implications for disease pathology.</p></div

Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis

Metastasis is a frequent and lethal complication of cancer. Vascular endothelial growth factor-C (VEGF-C) is a recently described lymphangiogenic factor. Increased expression of VEGF-C in primary tumours correlates with dissemination of tumour cells to regional lymph nodes. However, a direct role for VEGF-C in tumour lymphangiogenesis and subsequent metastasis has yet to be demonstrated. Here we report the establishment of transgenic mice in which VEGF-C expression, driven by the rat insulin promoter (Rip), is targeted to β-cells of the endocrine pancreas. In contrast to wild-type mice, which lack peri-insular lymphatics, RipVEGF-C transgenics develop an extensive network of lymphatics around the islets of Langerhans. These mice were crossed with Rip1Tag2 mice, which develop pancreatic β-cell tumours that are neither lymphangiogenic nor metastatic. Double-transgenic mice formed tumours surrounded by well developed lymphatics, which frequently contained tumour cell masses of β-cell origin. These mice frequently developed pancreatic lymph node metastases. Our findings demonstrate that VEGF-C-induced lymphangiogenesis mediates tumour cell dissemination and the formation of lymph node metastases

Trafficking of GAS visualized in dermal lymphatic vessels and draining cervical lymph nodes of the mouse ear pinna.

<p>A-C: Confocal microscopic images of murine ear skin 6–24 hours after local intradermal inoculation. (A) Low magnification images of lymphatic vessels in skin and surrounding infected tissue 24 hours post infection (scale bar 20 μm). (B) 3D rendered images and (C) orthogonal views of the same z-stacks at 6 hours post infection. Arrows indicate individual streptococci interacting with LYVE-1 dense regions of lymphatic vessel endothelium (scale bar 5 μm). D-E: Epifluorescence microscopic images of frozen sections of draining cervical nodes 6 hours post infection at low power (100X magnification)(D) and high power (400X)(E) (Scale bar 50 μm and 20 μm respectively). Red = LYVE-1, green = GAS, blue = nuclei. Arrows indicate encapsulated GAS adhering to clusters of LYVE-1.</p

LYVE-1 functional blockade reduces GAS dissemination to draining lymph nodes.

<p>Dissemination of M18 GAS in murine soft-tissue infection following LYVE-1 mAb blockade (n = 22/group). Quantitative culture of GAS at site of infection (A), ipsilateral draining LN (B), spleen (C) and blood (D) 3 hours post-infection. Lines depict median values in each case (Mann Whitney U;* = p<0.05, ** = p<0.01). (Control antibody group: circles indicate isotype control antibody and triangles indicate polyclonal control IgG).</p

Capsular HA is critical for GAS dissemination to lymphatics.

<p><b>(</b>A) Generation of isogenic acapsular strains of M18 and M89 GAS. Left-right; Comparison of bacterial growth by optical density (600 nm) and quantification of capsular HA expression expressed as fg of HA per colony forming unit. (B) Dissemination of wildtype and acapsular GAS in murine soft-tissue infection. Left to right; Bacterial burden (cfu) in ipsilateral lymph node and thigh 3 hours post-injection (n = 6 mice/group; line depicts median).</p