An examination of the proteolytic activity for bovine pregnancy-associated glycoproteins 2 and 12

Abstract

The pregnancy-associated glycoproteins (PAGs) represent a complex group of putative aspartic peptidases expressed exclusively in the placentas of species in the Artiodactyla order. The ruminant PAGs segregate into two classes: the 'ancient' and 'modern' PAGs. Some of the modern PAGs possess alterations in the catalytic center that are predicted to preclude their ability to act as peptidases. The ancient ruminant PAGs in contrast are thought to be peptidases, although no proteolytic activity has been described for these members. The aim of the present study was to investigate (1) if the ancient bovine PAGs (PAG-2 and PAG-12) have proteolytic activity, and (2) if there are any differences in activity between these two closely related members. Recombinant bovine PAG-2 and PAG-12 were expressed in a baculovirus expression system and the purified proteins were analyzed for proteolytic activity against a synthetic fluorescent cathepsin D/E substrate. Both proteins exhibited proteolytic activity with acidic pH optima. The k(cat)/K(m) for bovine PAG-2 was 2.7x10(5) m(-1) s(-1) and for boPAG-12 it was 6.8x10(4) m(-1) s(-1). The enzymes were inhibited by pepstatin A with a K(i) of 0.56 and 7.5 nm for boPAG-2 and boPAG-12, respectively. This is the first report describing proteolytic activity in PAGs from ruminant ungulates.

Figure 1. SDS-PAGE gels of expressed recombinant bovine PAGs-2 and 12 stained either with Coomassie blue or by Western blotting

Panel (A) shows Coomassie blue staining of fractions from a preparation of recombinant boPAG-2. Lane-1 contains the total proteins from the lysed insect cell pellet. Lane-2 contains the flow-through of lysate from the anti-FLAG column. Lanes 3–5 contain elution fractions of recombinant boPAG-2 in order of emergence from the column. Panels (B) and (C) show Western blot images of boPAG-2-containing fractions transferred from identically loaded gels and immuno-blotted with anti-PAG-2 polyclonal and anti-FLAG monoclonal antibodies, respectively. (D) Coomassie blue staining of fractions from a preparation of recombinant boPAG-12. Lane-1 contains total proteins from the insect cell lysate. Lane-2 contains the flow-through from the anti-FLAG column. Lanes 3–5 contain fractions in order of their elution from the column. (E) Western blot image of boPAG-12-containing fractions immunoblotted and detected with anti-FLAG monoclonal antibody.

Figure 2. The relative activity of boPAG-2 (triangles) and -12 (squares) zymogens as a function of pH

Each activity point is normalized by that protease’s maximum activity (relative fluorescence units) at its pH optimum. The error bars represent standard deviation in results obtained from duplicate reads from two separate experiments.

Figure 3. Activity measured after an immuno-precipitation assay performed on insect cell lysates

Anti-FLAG antibody was used as a capturing antibody and protein G matrix was used as a solid support. Immunoprecipitated proteins were reacted with the cathepsin D/E FRET substrate for one hour in pH 3.5 buffer. Liberated product was measured on a fluorescent plate reader.

Figure 4. Influence of pHon PAG-2 and PAG-12 activity

The pH-dependence of the activity of recombinant boPAG-2 (A) and boPAG-12 (B) before activation (squares) and following activation (triangles). The initial velocities (in relative fluorescent units/min) of each PAG against the fluorescent FRET-cathepsin D/E substrate at each pH were estimated from kinetic reads for 10 min.

Figure 5. Determination of kinetic parameters for PAG-2 and PAG-12

Progress curves global fitted with k_cat and K_m for digestion of cathepsin D/E substrate by recombinant boPAG-2 (A) and boPAG-12 (B) at pH 4.0 and 37°C. In (A), the FRET substrate and boPAG-2 concentrations for the upper and lower series are 1 μM substrate and 5 nM boPAG-2 enzyme and 0.2 μM substrate and 2.5 nM boPAG-2 enzyme, respectively. In (B), the substrate and boPAG-12 concentrations are 0.5 μM substrate and 39 nM boPAG-12 enzyme and 0.5 μM substrate and 17 nM boPAG-12 enzyme. The lines representing each progess curve are indicated in the panels.

Figure 6. Homology models to predict substrate binding site differences between bovine PAG-2 and PAG-12

Residues that distinguish the active sites of boPAG-2 (A) and boPAG-12 (B) and pepsin (not shown). The homology models were constructed by using the crystal structure of mature human pepsin bound to pepstatin (PDB code 1PSN) as template by using the SWISS-MODEL server (http://swissmodel.expasy.org//SWISS-MODEL.html) (Guex and Peitsch, 1997; Schwede et al., 2003; Arnold et al., 2006). The backbone ribbon progresses through the colors of the rainbow from blue at the N-terminus to red at the C-terminus. The conserved aspartate side chains critical for peptidase activity are red. Side chains of the active site cleft differing between boPAG-2 and -12 are plotted with standard atom colors of blue for nitrogen, red for oxygen, and green for carbon. The “flap” is light blue.