Purification and Subunit Characterization of the Rat Liver Endocytic Hyaluronan Receptor

Abstract

The endocytic hyaluronan (HA) receptor of liver sinusoidal endothelial cells (LECs) is responsible for the clearance of HA and other glycosaminoglycans from the circulation in mammals. We report here for the first time the purification of this liver HA receptor. Using lectin and immuno-affinity chromatography, two HA receptor species were purified from detergent-solubilized membranes prepared from purified rat LECs. In nonreducing SDS-polyacrylamide gel electrophoresis (PAGE), these two proteins migrated at 175-and ϳ300 kDa corresponding to the two species previously identified by photoaffinity labeling of live cells as the HA receptor (Yannariello-Brown, J., Frost, S. J., and Weigel, P. H. (1992) J. Biol. Chem. 267, 20451-20456). These two proteins co-purify in a molar ratio of 2:1 (175:300), and both proteins are active, able to bind HA after SDS-PAGE, electrotransfer, and renaturation. After reduction, the 175-kDa protein migrates as a ϳ185-kDa protein and is not able to bind HA. The 300-kDa HA receptor is a complex of three disulfide-bonded subunits that migrate in reducing SDS-PAGE at ϳ260, 230, and 97 kDa. These proteins designated, respectively, the ␣, ␤, and ␥ subunits are present in a molar ratio of 1:1:1 and are also unable to bind HA when reduced. The 175-kDa protein and all three subunits of the 300-kDa species contain N-linked oligosaccharides, as indicated by increased migration in SDS-PAGE after treatment with N-glycosidase F. Both of the deglycosylated, nonreduced HA receptor proteins still bind HA.HA 1 is an important extracellular matrix component of all tissues and plays a key role in development, cell proliferation, cell adhesion, recognition, morphogenesis, differentiation, and inflammation (1-4). The daily total body turnover of HA in humans is at least 1 g/day (4). HA degradation and removal in the body occurs via two clearance systems (3): one is in the lymphatic system, which accounts for ϳ85% of the HA turnover, and another system is hepatic, accounting for ϳ15% of the total body HA turnover. HA in tissues throughout the body is continuously synthesized and degraded. Very large HA molecules (ϳ10 7 Da) are partially degraded to large fragments (ϳ10 6 Da) that are then released from the matrix and flow with the lymph to lymph nodes. The majority of HA (ϳ85%) is completely degraded in the lymph nodes by unknown mechanisms and the remaining HA (ϳ15%) that passes through the nodes finally enters the blood. Clearance of this circulating HA is presumably important for normal health (3, 4). Elevated serum HA levels are found in several disease conditions such as liver cirrhosis, rheumatoid arthritis, psoriasis, scleroderma, and some cancers (5-7).LECs have a very active recycling, endocytic receptor that removes HA and other glycosaminoglycans, such as chondroitin sulfate, from the circulation (3, 8 -10). Earlier reports misidentified this LEC HAR as ICAM-1 (11, 12), also known as CD54, which is a 90-kDa protein. This finding was later recognized as an artifact in that ICAM-1 bound nonspecifica...

Expression, Processing, and Glycosaminoglycan Binding Activity of the Recombinant Human 315-kDa Hyaluronic Acid Receptor for Endocytosis (HARE)

“…The processing of the 315-HARE appears to be a tightly regulated process, since the ratio of 315-to 190-HARE was consistently ϳ3-4:1 in multiple experiments with the six cell lines used here. Earlier results also demonstrated a consistent ratio of the two isoforms in rat liver or human spleen and the possibility that isoform ratios might differ between tissues (30,33). From our protein sequencing data, the 190-HARE purified from human spleen begins at Ser 1136 .…”

“…Unlike the large native rat liver HARE isoform, which contains two large disulfide-bonded subunits of 220 and 250 kDa (33), the recombinant 315-kDa human HARE is a single polypeptide. By Western analysis, the 315-kDa protein band did not decrease in size after reduction with dithiothreitol, and multiple bands were not detected.…”

“…Whole cell lysates were subjected to reducing (R) or nonreducing (NR) conditions, separated by 5% SDS-PAGE, and electrotransferred to nitrocellulose as described under "Experimental Procedures." Nitrocellulose was cut into strips and blocked with TBST/BSA, and each strip was incubated with 1 g/ml mouse IgG or one of the eight indicated mAbs raised against the rat 175-kDa HARE (28,33). The 315-kDa HARE (filled arrows) and the 190-kDa HARE (open arrows) proteins were recognized by mAbs 30, 154, and 159 in the nonreduced samples, whereas both receptors reacted with only mAb 159 when reduced.…”

“…For example, the ratio of rat 175-HARE to 300-HARE in the liver is higher than in the spleen. Thus, the levels of each isoform appear highly regulated (30,33), but it is still not known why or how this occurs. We recently confirmed that the 190-kDa hHARE is, in fact, a functional endocytic recycling receptor that can mediate GAG uptake in the absence of the larger 315-kDa hHARE (37).…”

The Hyaluronan Receptor for Endocytosis (HARE) Activates NF-κB-mediated Gene Expression in Response to 40–400-kDa, but Not Smaller or Larger, Hyaluronans

“…The results confirm that HA binding to 190-hHARE is required for HARE-mediated NF-B-activated gene expression. Although we do not have a similar stable cell line expressing rHARE(⌬Link), previous studies showed that mAb-174, which was raised against rHARE, completely blocks HA uptake in stable cells expressing rHARE (29), in primary rat liver sinusoidal endothelial cells, and in intact perfused rat liver (30,49). mAb-174 does not recognize hHARE.…”