SHAP Potentiates the CD44-mediated Leukocyte Adhesion to the Hyaluronan Substratum

Lisheng Zhuo,Akiko Kanamori,Reiji Kannagi,Naoki Itano,Jiwen Wu,Michinari Hamaguchi,Naoki Ishiguro,Koji Kimata

doi:10.1074/jbc.m506703200

Lisheng Zhuo, Akiko Kanamori + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m506703200

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Abstract

CD44-hyaluronan (HA) interaction is involved in diverse physiological and pathological processes. Regulation of interacting avidity is well studied on CD44 but rarely on HA. We discovered a unique covalent modification of HA with a protein, SHAP, that corresponds to the heavy chains of inter-alpha-trypsin inhibitor family molecules circulating in blood. Formation of the SHAP.HA complex is often associated with inflammation, a well known process involving the CD44-HA interaction. We therefore examined the effect of SHAP on the CD44-HA interaction-mediated lymphocyte adhesion. Under both static and flowing conditions, Hut78 cells (CD44-positive) and CD44-transfected Jurkat cells (originally CD44-negative) adhered preferentially to the immobilized SHAP.HA complex than to HA. The enhanced adhesion is exclusively mediated by the CD44-HA interaction, because it was inhibited by HA, but not IalphaI, and was completely abolished by pretreating the cells with anti-CD44 antibodies. SHAP appears to potentiate the interaction by increasing the avidity of HA to CD44 and altering their distribution on cell surfaces. Large amounts of the SHAP.HA complex accumulate in the hyperplastic synovium of rheumatoid arthritis patients. Leukocytes infiltrated to the synovium were strongly positive for HA, SHAP, and CD44 on their surfaces, suggesting a role for the adhesion-enhancing effect of SHAP in pathogenesis.

Highlights

Documented in leukocyte extravasation and function in diverse systems
We showed in this study that the covalent association of SHAP with HA greatly enhanced its avidity to the cell-surface CD44 and provided a new insight into the complex regulatory mechanism for the CD44-HA interaction-mediated cell adhesion; that is, the regulation can occur on the HA side
There are some differences in the regulation pattern between SHAP and tumor necrosis factor-stimulated gene 6 product (TSG6)

Summary

MATERIALS AND METHODS

Antibodies, and Other Reagents—Human cutaneous T lymphoma cell Hut and human acute T leukemia cell Jurkat were purchased from ATCC. The SHAP1⁄7HA complex or HA in the sample solutions were captured on HABP (2 ␮g/ml in 0.1 M NaHCO3, pH ϳ9.5)-coated wells of MaxiSorp plates, and detected with rabbit anti-I␣I antibody and HRP-conjugated goat anti-rabbit IgG antibody, or biotinylated HABP and HRP-conjugated streptavidin, respectively. Immunolabeling and Fluorescence Microscopy—Hut cell suspension was laid on the SHAP1⁄7HA or HA-coated cover glass (prepared as mentioned above), followed by incubating at room temperature for 20 min or 1 h. The cover glass was blocked with 10% goat serum (Nichirei, Tokyo, Japan), incubated with PBS-T (0.1% Tween 20 in PBS) containing Ab4 antibody (1 ␮g/ml) and biotinylated HABP (0.5 ␮g/ml), and with PBS-T containing Alexa 594-rabbit anti-mouse IgG (1:1,000 dilution) and Alexa 488-streptavidin (1:1,000 dilution) at room temperature for 1 h each. A considerable population of cells exhibited a very low rolling speed, even stopping, on the SHAP1⁄7HA complex, in sharp contrast to those

RESULTS

Findings

DISCUSSION