For this, CLEC 2 e pres sion was induced on 293 T RE CLEC 2 cells, and bind ing of soluble podoplanin fused to the Fc portion of human immunoglobulin was analyzed by flow cytometry. Efficient binding of soluble podoplanin was observed only upon induced e pression of CLEC 2, and a control Fc protein did not bind to the CLEC 2 e pressing cells. Thus, 293T cells, which we and many others frequently use for production of HIV 1 stocks, e press podoplanin. and podoplanin specifically interacts with CLEC 2. Glycosylation of podoplanin is required for efficient binding to CLEC 2 We ne t sought to elucidate the determinants governing efficient interactions between podoplanin and CLEC 2. For instance, it is at present unclear if glycosylation of podoplanin is required for binding to CLEC 2.

Watson and colleagues demonstrated that binding of CLEC 2 to the snake venom protein rhodocytin is glycosylation independent, and defined several amino acids in CLEC 2 which contributed to efficient rhodocytin binding. Thus, mutations K150A, E187A, K190A and N192A decreased binding of CLEC 2 to rhodocytin in surface plasmon resonance binding studies. We addressed if these residues were also required for binding to soluble podoplanin. Flow cytometric analysis showed that all changes, with the e ception of K190A were com patible with efficient e pression of CLEC 2. Wild type CLEC 2 and all mutants, e cept K190A, bound to soluble podoplanin with similar efficiency, indicating that the CLEC 2 residues involved in rhodocytin binding were not important for binding to podoplanin.

Podopla nin contains sialylated O glycans, and we ne t ana lyzed if glycosylation of podoplanin is essential for binding to CLEC 2. For this, podoplanin Fc fusion pro teins were produced in wt CHO cells or CHO cells that due to defects in either the medial Golgi localized N acetylglucosaminyltransferase I or the trans Golgi localized CMP sialic acid transporter have lost their abilities to produce comple N glycans and sialylated glycoconjugates, respectively. Soluble proteins were concentrated from cellular supernatants by size e clusion filtration, and Western blot analysis showed that the podoplanin Fc preparations contained roughly comparable amounts of protein, while the Fc control protein preparation was more concen trated.

When binding to CLEC 2 was analyzed in a FACS based assay, podoplanin produced in Lec1 cells Brefeldin_A still bound to CLEC 2 with appreciable efficiency. In contrast, podoplanin produced in Lec2 cells and thus almost completely lacking sialoglycoconjugates did not show significant binding to CLEC 2. The observed differences indicate that the presence of sialic acid is essential for binding to CLEC 2. Moreover, because N glycans are e clusively of the high mannose type if proteins are e pressed in Lec1 cells, this finding provides evidence that sialylated O glycans are involved in mediating the contact to CLEC 2.