Figure 9.

Characterization of complexed form of HD-5 in ileostomy fluid of CD patient CD21. A: AU-PAGE Western blot analysis of RP-HPLC 59% acetonitrile fraction containing HD-5 from CD ileostomy fluid (Figure 8B). After AU-PAGE, the 59% acetonitrile fraction (lane 3), synthetic mature HD-5 (lane 2), and acid extract of ileal crypt epithelial cells (lane 1) were transferred to PVDF membrane for immunoblotting using anti-HD-5 antibody. In the 59% acetonitrile fraction of ileostomy fluid, a single HD-5-positive band was seen (lane 3), with electrophoretic mobility slower than that for pro-HD-5 in ileal crypt epithelial cell extract (lane 1). Amido black staining of the same membrane revealed a prominent protein band in the same position as the HD-5-immunoreactive band. N-terminal sequencing of this protein band revealed two sequences, IVNGEDAV and CGVPAIH, which match for chymotrypsinogen and chymotrypsin chain B, respectively. B: SDS-PAGE Western blot analysis of RP-HPLC 59% acetonitrile fraction containing HD-5 from CD ileostomy fluid (Figure 8B). After SDS-PAGE and transfer to PVDF membrane of the same samples as in A, immunoblotting showed that the HD-5-positive band now had electrophoretic mobility similar to that of synthetic mature HD-5. Amido black staining of the same membrane showed a prominent protein band at ∼27.5 kDa. Immunoblotting of a similar PVDF membrane using anti-chymotrypsin antibody also showed a prominent band at ∼27.5 kDa. C: Amino acid sequence of chymotrypsinogen (amino acids 19 to 263) and its signal sequence (amino acids 1 to 18). Cleavage for activation leads to chymotrypsin chain A (amino acids 19 to 33) and chymotrypsin chain B (amino acids 34 to 164). Sequences in bold were identified after ES-QToF MS analysis of HD-5-positive 59% acetonitrile fraction. Edman degradation revealed the underlined sequences. D: MS analysis of HD-5-positive 59% acetonitrile fraction revealed a mass of 3580 Da, consistent with predicted mass of mature HD-5 (see Figure 1).