B16 mouse melanoma cells adhere to and spread on laminin. We have previously shown that cell spreading is uncoupled from adhesion when unglycosylated laminin is used as a substratum; spreading was restored by a Pronase digest of laminin which became inactive when it was specifically depleted of its mannoside peptides; spreading was also specifically restored by mannosides such as mannan, Man9, and Man6, but not Man3. The effector mannosides bind to a cell surface receptor, previously shown by direct and indirect methods. We have now identified the receptor as cell surface calreticulin by isolating it via mannan affinity chromatography and showing its sequence identity with mouse calreticulin. Anti-calreticulin antibodies confirm this identity, decorate the B16 cell surface, and block cell spreading. Purified B16 cell calreticulin from whole cell lysates successfully competes with cell surface calreticulin and prevents cell spreading. The composite data implicate cell surface calreticulin as a putative lectin that must be occupied to initiate spreading of laminin-adherent B16 cells.
Several lines of evidence indicate that calreticulin has lectin-like properties. As a molecular chaperone, calreticulin binds preferentially to nascent glycoproteins via their immature carbohydrates; this property closely resembles that seen for calnexin, a chaperone with extensive molecular identity to calreticulin. A cell surface form of calreticulin also exhibits lectin-like properties, binding specific oligomannosides including those covalently linked to laminin. In the present study we examined the interaction between calreticulin and laminin by means of surface plasmon resonance. The results show that calreticulin specifically binds to glycosylated laminin but fails to specifically bind tunicamycin-derived unglycosylated laminin or bovine serum albumin. Calreticulin binding to glycosylated laminin requires calcium and is abolished in the presence of EDTA. Scatchard analysis of binding yields an apparent association constant, Ka, of 2.1 +/- 0.9 x 10(6) m-1 while kinetic analysis yields an estimate of the association on rate, (Kassoc), as 2 x 10(5) m-1 s-1. The composite results support calreticulin's lectin-like properties as well as its proposed role in laminin recognition, both in the cell interior and on the cell surface.
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