Matrix metalloproteinase-9 (MMP9)

MMP9 Protein Structure

Figure 1: MMP9 Protein Structure.

MMP9 Introduction

Protein Function

MMP9 is a secreted member of the zinc metalloproteinase family, also known as gelatinase B or 92 kDa type IV collagenase (EC 3.4.24.35). It is one of the two gelatinases in the matrix metalloproteinase (MMP) family. MMP9 is a multidomain protein with a molecular weight of 92 kDa, capable of catalyzing the degradation of large extracellular matrix (ECM) components such as elastin and collagen and specific cell surface proteins.
The precursor of MMP-9, containing the N-terminal propeptide PRCGXPD, is synthesized and secreted. The thiol group of cysteine in the N-terminal propeptide PRCGXPD chelates with the zinc in the active site, maintaining MMP-9 in its latent form. MMP-9's latent form primarily exists in two forms: as a monomer and as a homodimer linked by disulfide bonds.
During activation, the 92 kDa latent MMP9 undergoes two consecutive proteolytic cleavages. The first occurs at the Glu59-Met60 bond between propeptide helices 1 and 2, producing an 86 kDa intermediate form. Another cleavage occurs at residues Cys99 to Arg106-Phe107 downstream of the zinc coordination, generating an active form of around 82 kDa.
Prolonged exposure to 4-amino phenylmercuric acetate (APMA) or matrix metalloproteinase MMP3 further processes the 82 kDa MMP9 form, resulting in a final active product of approximately 67 kDa, lacking the C-terminal fragment or an inactive product of 50 kDa lacking the catalytic domain.
MMP9 degrades fibronectin but not laminin or Pz-peptide.
It may play a role in osteoclast absorption, cleaving KiSS1 protein at the Gly-|-Leu bond. It cleaves type IV and type V collagens into larger C-terminal three-quarters fragments and shorter N-terminal quarter fragments.
MMP9 may play a significant role in localized protein hydrolysis in the extracellular matrix and leukocyte migration.

Protein Expression

Normal alveolar macrophages and neutrophils produce it.
MMP9 is primarily produced by monocytes, inflammatory macrophages, neutrophils, and most cancer cells. However, MMP9 is explicitly stored in tertiary granules in neutrophils, from where it can be rapidly released. In other cell types, MMP9 is synthesized upon stimulation by cytokines.
MMP9 can be detected at the protein level in neutrophils.

Protein Localization

Extracellular matrix

Figure 2: ICC Experimental Result of MMP9 Protein. Anti-MMP9 [RM1020] Product (ab283575). Green: MMP9; Red: alpha Tubulin; Blue: DAPI

Figure 2: ICC Experimental Result of MMP9 Protein. Anti-MMP9 [RM1020] Product (ab283575). Green: MMP9; Red: alpha Tubulin; Blue: DAPI

Isoforms & Post-Translational Modifications

Human (P14780): 78 kDa (mature form, predicted), 92 kDa (pro-form, predicted)
Mouse (P41245): 80 kDa (mature form, predicted)
Rat (P50282): 78 kDa (mature form, predicted)
N- and O-glycosylation**

WB Experiment Tips

Precautions

MMP9 protein undergoes various post-translational modifications (PTMs) and different cleavage intermediate forms. Hence, the observed band size of MMP9 in Western blotting may not exactly match the predicted protein size.
MMP-9 expression levels are low in many cell lines, making detecting it challenging. Various reagents can induce
MMP-9 expression, such as inflammatory factors, growth factors, and TPA (12-O-tetradecanoylphorbol-13-acetate). PMA can induce MMP-9 protein expression in cells, and specific protocols can be found on the official website here.
Setting a positive control is crucial due to the selective expression of MMP9 in specific tissues and cells.
Since MMP9 is a secretory protein and may not be detectable in cells, consider enriching protein detection in cell supernatants.

Positive Controls:

Whole cell lysate of U-2 OS cells treated with 200 nM TPA for 24 hours.
Lung tissue lysate from humans or mice.

Negative Controls (No or Low Expression):

Whole cell lysate of U-2 OS cells.
Whole cell lysate of RAW 264.7 cells.

Example Results

WB with Anti-MMP9 [RM1020] antibody (ab283575)

Figure 3: WB with Anti-MMP9 [RM1020] antibody (ab283575).

Primary Antibody: Anti-MMP9 antibody used at a dilution of 1/1000.

Lane 1: Whole cell lysate of U-2 OS cells.
Lane 2: Whole cell lysate of U-2 OS cells treated with 200nM TPA for 24 hours.
Lane 3: Whole cell lysate of RAW 264.7 cells.
Lane 4: Whole cell lysate of RAW 264.7 cells treated with 100ng/ml LPS for 4 hours followed by 1ug/ml BFA for 3 hours.
Lane 5: Whole cell lysate of NR8383 cells.
Lane 6: Whole cell lysate of NR8383 cells treated with 100ng/ml LPS for 4 hours followed by 1ug/ml BFA for 3 hours.
Lane 7: Lung tissue lysate from humans.
Lane 8: Lung tissue lysate from mice.

Predicted Band Size: 78 kDa
Detected Band Size: 84-92 kDa

Key control points

In addition to the routine issues that need to be paid attention to in the experiment, special attention should be paid to the following critical control points:

Sample preparation:

Use a combination of protease inhibitors to prevent the target protein from being degraded.
Keep the sample on ice during the entire sample preparation process.
Determine the total protein concentration of the sample by Bradford analysis, Lowry analysis, or BCA analysis.
Set up positive and negative controls.

Electrophoresis:

Load at least 20μg of total protein for electrophoresis.

Transfer:

It is strongly recommended to use Ponceau red staining after transfer to determine whether the transfer is successful.