VII The LYVE1 and HARE Receptors for HA Endocytosis and Degradation
Throughout the body, HA is continuously synthesized and degraded, and the turnover of HA in mammalian tissues has been extensively studied (5,196-198).
HA levels in the extracellular milieu are carefully modulated, as the level of free HA influences a number of hydrodynamic systems in the organism, such as lung fluid balance (199). High molecular mass HA is synthesized in the plasma membrane of fibroblasts and other cells by the addition of sugars to the reducing end of the growing polymer chain, with the non-reducing end protruding outside the cell (200,201). The rate of synthesis of HA by cells can be affected by cell density (202), mechanical stretch (26), and by growth factors, including transforming growth factor b, EGF and insulin-like growth factor 1 (203,204).
The turnover rate of HA is comparatively rapid for a connective tissue matrix component (tj=2 0.5 to a few days) (5). The lymphatic system accounts for 85% of the HA degradation in the body and the remaining 15% is degraded via the liver (5,196-198,205-209). Degradation of HA in the ECM occurs in two phases. Large native HA molecules (~ 10,000 kDa in the ECM) are partially degraded to fragments of ~ 1000 kDa (5). The final HA degradation process involves uptake of free HA released from the matrix into lymphatic vessels, followed by delivery to lymph nodes and finally to liver sinusoids for terminal hydrolysis (5,206,209). Six hyaluronidase-like genes have been identified in the human genome which takes part in HA degradation (210). Hyaluronidase-1 (Hyal-1) is found in mammalian plasma and urine, and also in major organs such as liver, kidney, spleen and heart (210). Data suggest that Hyal-1 and Hyal-2 are the major mammalian hyaluronidases in somatic tissues, acting in concert extracellularly to degrade HMW HA to the tetrasaccharide (210,211). In liver endothelial cells and Kupffer cells, experiments have determined that substantial levels of enzymes are present to fully degrade HA. Nine enzymes, including b-D-glucuronidase, b-N-acetyl-D-hexosaminidase and N-acetylglucosamine-6-phosphate deacetylase were shown to be present in endothelial cells and Kupffer cells, but were completely absent from hepatocytes, supporting the hypothesis that endothelial and Kupffer cells are primarily responsible for the final degradation of HA in the liver (211).
The lymphatic vessel endothelial HA receptor (LYVE-1) was first identified as the primary protein responsible for HA uptake in the lymph endothelium, and was found based on its cDNA homology (43%) with the HA receptor CD44 (212). Based on its sequence and structure, LYVE-1 was placed along with CD44 in the Link protein superfamily (36,212). LYVE-1 is also expressed in the sinusoidal endothelium of liver and spleen, the sites where uptake and degradation of HMW HA is known to occur (213). LYVE-1 is a 322 residue type I transmembrane glycoprotein, and can bind both immobilized and soluble HA (36,212). Despite similarities between the LYVE-1 and CD44-binding regions for HA, it is likely that LYVE-1 preferentially binds larger HA fragments than those recognized by CD44, probably >HA6 (36). LYVE-1 does not contain sequence homology with the CD44 transmembrane or cytoplasmic domains (36); these domains are responsible for the interaction of CD44 with the cytoskeleton and other signaling molecules. Thus far, similar interactions for LYVE-1 have not been identified.
In addition to LYVE-1, the HARE protein is also believed to endocytose HA for terminal degradation (214,215). HARE was first identified in liver endothelial cells, and is distributed in liver sinusoids, venous sinuses of the red pulp in the spleen and the medullary sinuses of lymph nodes (215). Although the sinusoidal liver and lymph endothelial cells also express CD44, antibodies directed against CD44 did not block HA internalization and degradation (216).
Like LYVE-1, HARE is a HA-binding type I membrane protein. HARE was identified as a member of the protein family of fasciclin, a primarily alpha-helical, lipid-linked cell-surface glycoprotein that can act as a homophilic adhesion molecule in tissue culture (214,217,218). Two forms of HARE have been identified, a 175 and a 300 kDa forms; the 175 kDa form is a monomer, but the 300 kDa form is a trimer, made up of alpha, beta and gamma subunits, at 260, 230 and 97 kDa, respectively (214,215). Based on monoclonal antibody cross-reactivity, it was determined that the 175 HARE monomer was related to the 260 and 230 kDa subunits of the 300 kDa form (215,219,220). Complete inhibition of HA internalization and subsequent degradation requires the simultaneous blocking of both the 300 and 175 kDa HARE proteins, although the two receptors appear to function independently (217,221)
Internalization of the HA-HARE complex occurs via clathrin-coated pit-mediated endocytosis in a Ca2+-independent manner (219,222,223). HARE was substantially co-localized with clathrin in cells, but not with internalized HA that was delivered to lysosomes (217). In a mechanism which corresponds to many other cell surface receptors, once HARE has delivered HA to pre-lysosomal compartments for transport to lysosomes, HARE itself is recycled to the cell surface to be used for further endocytosis cycles (224,225). Internalization of HA by HARE was not affected by treatment of cells with the cytokines tumor nectrosis factor a, interferon g or interleukin 1, or by treatment with Escherichia coli endotoxin (226). The direct interactions of HARE with cytoplasmic protein have not been determined; this issue, along with the signaling mechanisms directing the cellular uptake of HA, remains as topic for further research.
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