Heterogeneity of serum gelatinases MMP‐2 and MMP‐9 isoforms and charge variants

Abstract

The matrix metalloproteinases (MMPs) gelatinase A (MMP-2) and gelatinase B (MMP-9) are mediators of brain injury in multiple sclerosis (MS) and valuable biomarkers of disease activity. We applied bidimensional zymography (2-DZ) as an extension of classic monodimensional zymography (1-DZ) to analyse the complete pattern of isoforms and post-translational modifications of both MMP-9 and MMP-2 present in the sera of MS patients. The enzymes were separated on the basis of their isoelectric points (pI) and apparent molecular weights (Mw) and identified both by comparison with standard enzyme preparations and by Western blot analysis. Two MMP-2 isoforms, and at least three different isoforms and two different states of organization of MMP-9 (the multimeric MMP-9 and the N-GAL-MMP-9 complex) were observed. In addition, 2-DZ revealed for the first time that all MMP-9 and MMP-2 isoforms actually exist in the form of charge variants: four or five variants in the N-GAL complex, more charge variants in the case of MMP-9; and five to seven charge variants for MMP-2. Charge variants were also observed in recombinant enzymes and, after concentration, also in sera from healthy individuals. Sialylation (MMP-9) and phosphorylation (MMP-2) contributed to molecular heterogeneity. The detection of charge variants of MMP-9 and MMP-2 in MS serum samples illustrates the power of 2-DZ and demonstrates that in previous studies MMP mixtures, rather than single molecules, were analysed. These observations open perspectives for better diagnosis and prognosis of many diseases and need to be critically interpreted when applying other methods for MS and other diseases.

Matrix metalloproteinase activation by free neutrophil elastase contributes to bronchiectasis progression in early cystic fibrosis

“…The appearance of an additional ∼170-kDa band may be due to activation of a ∼220-kDa dimer MMP-9 by neutrophil elastase. Three BALF samples exhibited similar bands with slightly lower migration mobility, as described elsewhere [24]. Gelatinolytic bands ∼65 kDa in size ( fig.…”

Disposable MMP-9 sensor based on the degradation of peptide cross-linked hydrogel films using electrochemical impedance spectroscopy

“…This biomarker correlates with MRI studies in relapsing-remitting multiple sclerosis (RR MS) (Waubant et al, 1999). MMP-9 is present in RR MS as an inactive form (92 kDa) and active form (82 kDa and 65 kDa) (Rossano et al, 2014). Numerous studies show elevated levels of MMP-9 in people with RR MS.…”

Anti-inflammatory nutritional intervention in patients with relapsing-remitting and primary-progressive multiple sclerosis: A pilot study

“…Among the two gelatinases, the 82 kDa isoform is the first to increase in case of MS worsening. 46 This is understandable, as the 82 kDa MMP-9 can still be inhibited by its natural inhibitor tissue inhibitor of metalloproteinase-1 (TIMP-1), whereas the 65 kDa MMP-9 is TIMP-1 resistant and is the predominant active MMP-9 isoform in the course of MRI activity in MS. 74 The disease worsening observed in eight out of the 29 patients with dietary prescription, might be a matter of both the short treatment duration and inter-individual differences of gut dysbiotic microbiota to change in response to dietary intervention. …”

“…The ratio was first calculated for each individual patient (within each group), and then averaged for each group at various times 82 kDa and the 65 kDa MMP-9. This latter can be distinguished from MMP-2 by separation through concanavalin A 47 or by 2-DZ, 46 not shown here. However, in routine assay, 1-DZ is sufficient to provide useful data to assess the presence of activated MMP-9 isoforms, long before that clinical signs or RMI evidences appear.…”

“…46 1D zymography (1-DZ) was carried out as follows. Aliquots of sera containing 25 mg of proteins were supplemented with 15 mL of the non-reducing electrophoresis loading buffer (4% SDS, 12% glycerol, 0.01% bromophenol blue, 50 mmol/L Tris-HCl at pH 6.8) and analyzed in 8% polyacrylamide gel copolymerized with 0.1% (w/v) gelatin.…”