Fig. 1. Identification and characterization of HD6 in intestinal tissue and fluid. (a) Left: primary amino acid sequences of proHD6 and HD6. The pro region is in red, the sequence of mature HD6 is in purple, and the disulfide linkages are depicted as black lines. The underlined amino acid sequences are matched with the results from Edman degradation shown in (c) and (d). Right: the crystal structure of mature HD6 (PDB ID: 1ZMQ).12 The disulfide bonds are shown in yellow. (b) Immunogold labeling transmission electron microscopy of human small intestinal tissue. (i) Left panel: low magnification of Paneth cell granules as a negative control where primary antisera was omitted, scale bar = 2 μm. (ii) Center-left panel: immunogold double-labeling of Paneth cell granules for HD5 (5 nm gold particles) and HD6 (15 nm gold particles), scale bar = 500 nm, (iii) center-right panel: immunogold labeling of a single Paneth cell granule demonstrating co-packaging of HD5 (5 nm gold particles, open arrowheads) and HD6 (15 nm gold particles, filled arrowheads), scale bar = 200 nm. (iv) Right panel: immunogold double-labeling of Paneth cell granules for HD5 (5 nm gold particle, open arrowheads) and trypsin (15 nm gold particles, filled arrowheads), scale bar = 100 nm. (c) and (d) Analysis of HD6 in human small intestinal tissue (c) and luminal fluid (d) by Western blot, mass spectrometry, and N-terminal Edman degradation. Tissue extracts of human ileum and ileal fluid aspirates were resolved by AU-PAGE, transferred onto a PVDF membrane, and probed for HD6. Two separate gels with mobility normalized according to migration of tracking dye to gel bottom are shown and reveal HD6 immunoreactivity in both samples.
