N-TERMINAL AND CENTRAL REGIONS OF THE HUMAN CD44 EXTRACELLULAR DOMAINPARTICIPATE IN CELL-SURFACE HYALURONAN-BINDING

CD44 molecules are cell surface receptors for hyaluronan (HA). To defi ne regions of the extracellular domain of CD44 that are important for HA binding, we have studied the ability of HA-blocking CD44 mAbs to bi nd to CD44 from a variety of sources. Five CD44 mAbs (5F12, BRIC235, 3 F12, BU-75, and HP2/9) of 21 studied were identified that at least par tially blocked FITC-labeled HA (HA-FITC) binding to the standard form of CD44 (CD44S) in CD44-transfected jurkat cells. Analysis of reactivi ty of HA-blocking CD44 mAbs defined three distinct epitopes. Lack of r eactivity of mAb 5F12 with a CD44 fusion protein (CD44-Rg) containing an N-terminal truncation of 20 amino acids (aa), as well as reactivity of mAb 5F12 with an N-terminal CD44 synthetic peptide (CD44-9A), demo nstrated that the N-terminal proximal region of CD44 (aa 1 to 20) was involved in mAb 5F12 binding. A mutant cell line, CEM-NKR, derived fro m the T-ALL cell line, GEM, did not bind mAb 5F12 nor bind HA, whereas wild-type CEM did bind mAb 5F12 and HA. Sequence analysis of wild-typ e CEM and CEM-NKR CD44 cDNA demonstrated a G to A point mutation at po sition 575 in the CD44 cDNA of CEM-NKR, resulting in an arginine to hi stidine mutation at aa position 154. Taken together, our studies demon strated that there are three epitopes to which HA-blocking mAbs bind i n the extracellular domain of CD44, and that the CD44 N-terminal proxi mal and central regions are two regions in the extracellular domain of CD44 that may interact and either mediate or regulate HA binding to c ell surface CD44.