Title : Reducing Macro- and Microheterogeneity of
N-Glycans Enables the Crystal Structure of the Lectin and
EGF-Like Domains of
Human L-Selectin To Be Solved at 1.9 Å Resolution
Abstract :
- L-Selectin , a cell-adhesion receptor on the surface of most leukocytes, contains seven N-glycosylation sites
- In order to obtain the crystal structure of human L-selectin , we expressed a shortened version of L-selectin comprising the C-type lectin and EGF-like domains (termed LE) and systematically analysed mutations of the three glycosylation sites ( Asn22, Asn66 and Asn139 ) in order to reduce macroheterogeneity
- After we further removed microheterogeneity, we obtained crystals that diffracted X-rays up to 1.9 Å from a variant (LE010) with exchanges N22Q and N139Q and one GlcNAc2 Man5 N-glycan chain attached to Asn66
- Crystal-structure analysis showed that the terminal mannose of GlcNAc2 Man5 of one LE010 molecule was coordinated to Ca2 + in the binding site of a symmetry-related LE010
- The orientation of the lectin and EGF-like domain was similar to the described "bent" conformation of E- and P-selectins
- The Ca2 + -binding site reflects the binding mode seen in E- and P-selectin structures co-crystallised with ligands