Figure 1.
Peptides stimulate PSA activity towards small peptide substrates.
Peptide C4 stimulated the activity of PSA (mean ± SD) more efficiently than peptide B2-NH2 towards (A) a chromogenic peptide substrate (n = 2) and (B) a fluorogenic peptide substrate (n = 3). Dashed lines indicate the activity of PSA without peptide stimulation.
Table 1.
Enzyme kinetics of PSA with small peptide substrates.
Table 2.
Stimulation of PSA activity towards small peptide substrates with peptides B2-NH2 and C4.
Figure 2.
Degradation of nidogen-1 by PSA.
PSA cleaved both mouse nidogen-1 in Matrigel and human recombinant nidogen-1. Mass spectrometry analysis identified nidogen-1 in the silver stained gel bands (arrows, left panel). Nidogen-1 bands of 140 kDa (A) and 110 kDa (B) disappeared and fragments of 90 kDa (C) and 55 kDa (D) appeared after 48 h incubation of diluted Matrigel with PSA at 37°C. Nidogen-1 cleavage by PSA in Matrigel was visualized by Western blotting with anti-nidogen-1 polyclonal antibody (middle panel). PSA (1 µM) cleaved recombinant human nidogen-1 (0.5 µM) into two fragments 85 kDa (arrow with *) and 55 kDa (arrow with **) during 20 h incubation at 37°C (right panel).
Figure 3.
Cleavage of protein substrates by PSA.
(A) Characterization of the proteolytic activity of PSA (0.2 µM) during 22 h incubation towards different protein substrates (1 µM each, except 0.5 µM semenogelin I) by SDS-PAGE and silver staining. Approximate molecular weights of the proteins are: PSA (28 kDa), semenogelin I (50 kDa), semenogelin II (63 kDa), fibronectin (220 kDa), galectin-3 (26 kDa), IGFBP-3 (30 kDa) and nidogen-1 (130 kDa). The lanes in which ∼50% of the proteins were cleaved are bordered. (B) 1 µM MMP-3 (22 kDa), but not PSA, cleaved 1 µM plasminogen (88 kDa). Also 0.5 M fibronectin was incubated with 1 µM PSA as a control (SDS-PAGE with silver staining).
Figure 4.
The effect of peptides on the proteolytic cleavage of different protein substrates by PSA.
(A) Peptide B2 enhanced the activity of PSA towards all protein substrates more strongly than C4, except for fibronectin this could not be detected. The peptides were preincubated with 0.2 µM PSA for 30 min prior to addition of 0.3–2.5 µM protein substrates and the cleavage of the proteins was detected after 10 min (semenogelin I), 40 min (semenogelin II), 4 h (fibronectin and nidogen-1) and 20–22 h (galectin-3 and IGFBP-3) incubation at 37°C. The notation 5* indicates that 5 µg of either peptide (with semenogelins I and II peptide C4, and with the other proteins B2) was added to the control sample. (B) Concentration of intact IGFBP-3 after incubation with PSA and peptides shown in relation to IGFBP-3 control without added PSA as measured by immunofluorometric assay that recognizes only intact, not cleaved IGFBP-3 (n = 2, mean ± SD).
Figure 5.
HUVEC tube formation assay with nidogen-1 and galectin-3.
Nidogen-1 and galectin-3 or their PSA-generated fragments did not have any effect on tube formation. Control wells are with PBS and PSA. Images are representative examples from two separate experiments.