Ubiquitin-Like Protein from Human Placental Extract Exhibits Collagenase Activity

<div><p>An aqueous extract of human placenta exhibits strong gelatinase/collagenase activity in zymography. 2-D gel electrophoresis of the extract with gelatin zymography in the second dimension displayed a single spot, identified as ubiquitin-like component upon MALDI/TOF MS/MS analysis. Immunoblot indicated presence of ubiquitin and absence of collagenase in the extract. Collagenase activity of the ubiquitin-like component was confirmed from the change in solubility of collagen in aqueous buffer, degradation of collagen by size-exclusion HPLC and atomic force microscopy. Quantification with DQ-gelatin showed that the extract contains 0.04 U/ml of collagenase activity that was inhibited up to 95% by ubiquitin antibody. Ubiquitin from bovine erythrocytes demonstrated mild collagenase activity. Bioinformatics studies suggest that placental ubiquitin and collagenase follow structurally divergent evolution. This thermostable intrinsic collagenase activity of placental extract might have wide physiological relevance in degrading and remodeling collagen as it is used as a drug for wound healing and pelvic inflammatory diseases.</p> </div

AFM of collagen.

<p>(A) Enlarged 3D view of collagen monomer filament (diameter 82±10 nm). (Lower Panel) Cross-section indicating intact collagen filament. (B) Amplitude flattened image of collagen (67.7 nm) incubated with collagenase. A fiber (7 nm) appearing from its side is also apparent. The bulge diameter of 32 nm is similar to another bulge (∼28.7 nm). (Lower Panel) Loss in integrity of collagen. The arrow on the left indicates the 7 nm fiber while that on the right indicates the 67.7 nm fiber. (C) Amplitude flattened image of collagen incubated with peptide fraction indicated two fragments of lengths 598 and 475 nm and width 59.6 nm each. (Lower Panel) Loss in integrity and fragmentation of collagen. (D) Topograph flattened image of collagen treated with ubiquitin indicates a single particle with a bulging head; scattered bulges are also apparent. (Lower Panel) Loss in integrity of collagen. The arrow indicates poor peak shape of the collagen and the possible region of wearing out from this molecule. In all the figures presented, a color scale bar indicating sample height from the mica sheet has been represented. The dark shade indicates depression of the sample while the white color represents maximum elevation.</p

Identification of the component responsible for collagenase activity.

<p>(A) 2-D gel electrophoresis of placental extract (100x). Positions of Mw markers are indicated. (B) Gelatin zymography in the second dimension. The activity corresponded to spot number 3 of A. (C) Immunological cross-reactivity between anti-sera of ubiquitin and (a) ubiquitin, (b) peptide fraction and (c) ovalbumin. (D) Immunological cross-reactivity between anti-sera of peptide fraction and (a) collagenase, (b) peptide fraction and (c) ovalbumin. (E) Gelatin zymography. Lane 1, ubiquitin (bovine); and Lane 2, peptide fraction. The arrow indicates the faint band of ubiquitin which roughly corresponds to that of placental ubiquitin. (F) Silver stained SDS-PAGE of ubiquitin (bovine, lane 1) and peptide fraction (lane 2). Approximate Mw of ubiquitin bands have been indicated.</p

Comparison between ubiquitin and collagenase.

<p>(A) 2.0.12 ClustalW Sequence alignment between human ubiquitin and 1C3TA (mutant ubiquitin). The ‘*’ and ‘:’indicates identical and highly similar residues respectively. The mutated sequences have been highlighted in bold-red, which are also highly similar to corresponding ubiquitin amino acid residues. (B) Multiple sequence alignment of the peptidase domains of the matrix metalloproteases, obtained from their corresponding crystal structures, with 1C3TA by MAFFT (71) shows conservation of E₅₁ in 1C3TA with the catalytic E of the MMPs. The sequences have been represented as the MMP family_PDB ID. (C) Structural superimposition of the 3D-coordinates of the peptidase domain with the crystal structure of 1C3TA (I). Superimposition of 1AYK, of MMP1, peptidase domain (green) with 1C3TA (cyan). Panel II. Superimposition of 1A85, of MMP8, peptidase domain (green) with 1C3TA (cyan). Panel III. Superimposition of 1EUB, of MMP13, peptidase domain (green) on 1C3TA (cyan).</p

Gelatinase/Collagenase activity of Placental Extract.

<p>(A) Gelatin Zymography - Lane 1, MMP 2; lanes 2, 4 and 6, MMP 2 activation profile in presence of APMA for 0, 1 and 2 hrs; lanes 3, 5 and7, MMP 2 incubated with peptide fraction at 37°C for same duration. (B) Gelatin Zymography - Lane 1 and 2, 0.1 ng collagenase incubated at pH 7.5 for 0 and 20 h at 37°C; lanes 3 and 4, 20 µg peptide fraction with 0.1 ng bacterial collagenase incubated for 0 and 20 h. (C) Collagen zymography. Lanes 1 and 2, 1.0 and 0.1 ng of collagenase; Lane 3, 20 µg of placental peptide fraction. (D) SE-HPLC analysis of collagenase activity of placental extract. a: collagen (R_t = 9.66±0.05 min, n = 5), b: placental extract (15.46±0.08 min), c : collagen treated with the extract at 0 h (major components at 7.3±0.03 and 16.1±0.08 min) and d : 168 h (major components at 7.56±0.01, 8.20±0.03, 8.83±0.001 and 9.60±0.04 min) respectively (n = 5).</p