Fig 1: Allosteric interaction domain of MMP9 and oligonucleotide.(A) Cartoon illustration of the domain structure of MMP9. Amino acid residues are indicated for the tripartite FNII repeat followed by the enzymatic cleft co-ordinated with the catalytic zinc molecule within the catalytic domain. Signal peptide, SP; cysteine switch, C; O-glycosylation domain, OG. (B) Dose–response of enzymatic potentiation for the purified full-length (closed circle, solid line) or purified catalytic domain (open circle, dashed line) of MMP9. The catalytic domain spans amino acid residues 107–449 as indicated in the cartoon above. (C) Enzymatic activity of overexpressed MMP9.mCherry without (white) or with 1 µM ssDNA (gray) of full-length (FL) control or different truncation/deletion mutants. mCherry fluorescence is used to normalize MMP9 input. Hemopexin domain truncation, tHmPx; O-glycosylation domain and hemopexin domain truncation, tOG; triple fibronectin-domain deletion, ΔFNII (Δ1–3); double fibronectin-domain deletion, ΔFNII (Δ2–3); single fibronectin-domain deletion, ΔFNII (Δ3). Domain 1 contains residues 224–271, domain 2 contains residues 282–329, and domain 3 contains residues 341–388. Western blots of mCherry-tagged MMP1, MMP2, MMP3, or MMP9 (no 6xHis tag) expressed in transfected HEK293 cells detected by blotting with an anti-mCherry antibody. Cellular lysate (D) or culture media (E) show expressed proteins of the expected molecular mass. (F) Relative expressed protein amounts in the culture media by direct measurement of mCherry fluorescence. (G) MMP activity assay normalized by mCherry fluorescence without (white) or with (gray) 1 µM ssDNA.
Fig 2: Oligonucleotide-induced enhanced PC12 neurite outgrowth.(A) Immunofluorescent labeling of PC12 neurites in media only (left), NGF stimulation (middle), or NGF and ssDNA stimulation (left). β-III-Tubulin (red) was used to stain the cytoplasm and neurite extensions, while Hoechst stain (blue) was used for nuclear staining. Scale bar = 50 µm. (Ai) Neurite Tracer software was used to quantify the number of nuclei and (Aii) the total cytoplasm/neurite density. (Aiii) Neurite density per cell was calculated by normalizing the neurite density by total number of nuclei. (Aiv) Prior to immunofluorescent labeling, culture medium was removed and MMP enzymatic activity was measured. (B) Immunofluorescent labeling of sgBlank control (left) and MMP9 KO PC12 cells (right) in media only (top), NGF stimulation (middle), or NGF and ssDNA stimulation (bottom) with Hoechst nuclear stain on the left, α-III-tubulin in the middle, and merged analysis on the right. Scale bar = 100 µm. (C) Neurite density per cell was calculated for each stimulation condition in wild-type (sgBlank) or MMP9 KO PC12 cells. (D) MMP enzymatic activity of conditioned media for each condition in wild-type (sgBlank) or MMP9 KO PC12 cells.
Fig 3: Interaction of DNA and RNA oligomers with MMP9.(A) PCR amplification of DNA oligomers co-purified with either MMP9.mCherry.His or mCherry.His. (B) Increasing concentrations of purified MMP9 catalytic domain or OVA were incubated with ssDNA oligomers. Protein : DNA interactions were analyzed by an electrophoretic mobility shift on an agarose gel. (C) Fluorescence polarization of FAM-labeled ssDNA incubated with MMP9 (full length) or OVA. Saturating levels of ssDNA-Biotin were used for fluorescence polarization competition of ssDNA-FAM. (D) Fluorescence polarization of increasing concentrations of 40-mer RNA or 40-mer ssDNA incubated with MMP9 or OVA. Dose–SPR response of increasing 40-mer ssDNA oligomer bound to MMP9.mCherry.His (E) or mCherry (F).His-labeled NTA-Ni-SPR flow cells. The traces are for 100, 30, 10, 3, 1, and 0.3 µM ssDNA.
Fig 4: Nucleic acid potentiation of MMP9 enzymatic activity.(A) Real-time analysis of cleaved fluorescent-labeled MMP substrate of MMP9 only (open circle, solid line), DNA oligomer only (black circle, dashed line), or MMP9 and 40-mer DNA (black circle, solid line). (B) Fold induction of enzymatic activity above MMP9 only with 40-mer ssDNA, 40-mer dsDNA, or 40-mer RNA (all at 1 µM). dsDNA was confirmed by ethidium bromide staining on agarose gel (inset), ssDNA and RNA showed low-level ethidium bromide intercalation due to some intramolecular ds character. (C) Fold induction over MMP9 alone of different length DNA oligomers or dNTPs. All nucleotides were present at 1 µM in the activity assay reaction. (D, top) A surface electrostatic potential of C-terminus truncated MMP9 crystal structure (PDB: 1l6j) displayed using PyMOL. Red indicates positive and blue indicates negative charges. (Bottom) Fold induction of MMP9 enzymatic activity with increasing concentrations of different polyanionic molecules over MMP9 activity alone. (E) Western blot detection of MMP9-induced PARP-1 cleavage fragments using a polyclonal antibody (top). Densitometric analysis of the different cleavage fragments of PARP-1 alone (white), PARP-1 with MMP9 (gray), and PARP-1 with MMP9 and increasing concentrations of ssDNA (gray with dashes).
Supplier Page from Enzo Life Sciences, Inc. for MMP-9 (catalytic domain) (human), (recombinant, E. coli)