Figure 2. CD99 lung cancer-associated mutations of Asp92 lead to a shift of the meprin β cleavage site.

(A) Schematic diagram of CD99 processing at the cell surface. Meprin β cleaves the full-length CD99 protein within highly conserved regions (HCR) II and III creating C-terminal fragments (CTF) I and II, respectively. CTF II is further processed by γ-secretase thereby releasing an intracellular domain (ICD) into the cytosol. For all further experiments, a C-terminally myc-/FLAG-tagged construct was used. HCRs are indicated by dark blue boxes with white Roman numerals. Arrowhead indicates meprin β cleavage site between Ser91 and Asp92 (red) affected by the mutations. C, C-terminus. N, N-terminus. (B) Genomic location of the lung cancer-associated CD99 patient mutations. The human CD99 gene (red) is located in the pseudoautosomal region (PAR) 1 at the end of the short arm of the X chromosome (blue). The mutations c.274G>C and c.274G>T (situated in exon 6) cause amino acid exchanges from Asp92 to His92 and Tyr92, respectively, thereby leading to loss of negative charge at the P1’ position, which is preferred by meprin β. p, p arm. q, q arm. (C) RP-HPLC chromatogram of synthetic peptides representing CD99 WT, D92H, and D92Y. Peptides spanning the meprin β cleavage site around Asp92 were analyzed alone (red graph) or after incubation with 15 nM recombinant meprin β for 1 h (black graph). Collected elution fractions were analyzed by MALDI-TOF MS and several cleavage sites were identified within the peptide sequences (black arrowheads). AU, arbitrary units. (D) Quantification of the decreasing FL peak areas from (Supplementary Figure 3). (E) Quantification of the increasing cleavage product peak areas from (Supplementary Figure 3).