An SNP variant MT1-MMP with a defect in its collagenolytic activity confers the fibrotic phenotype of Dupuytren’s Disease

  1. Yoshifumi Itoh
  2. Michael Ng
  3. Akira Wiberg
  4. Katsuaki Inoue
  5. Narumi Hirata
  6. Katiucia Batista Silva Paiva
  7. Noriko Ito
  8. Kim Dzobo
  9. Nanami Sato
  10. Valentina Gifford
  11. Yasuyuki Fujita
  12. Masaki Inada
  13. Dominic Furniss

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Abstract

Dupuytren’s Disease (DD) is a common fibroproliferative disease of the palmar fascia. We previously identified a strong association with a non-synonymous variant (rs1042704, pD273N) in MMP14 (encoding MT1-MMP). We investigated the functional consequences of this variant, and demonstrated that the variant MT1-MMP (MT1-N 273 ) exhibits only 17% of cell surface collagenolytic activity compared to the ancestral enzyme (MT1-D 273 ). Cells expressing both MT1-D 273 and MT1-N 273 in a 1:1 ratio, mimicking the heterozygous state, possess 38% of the collagenolytic activity compared to the cells expressing MT1-D 273 , suggesting that MT1-N 273 acts in a dominant negative manner. Consistent with this hypothesis, patient-derived DD myofibroblasts expressing MT1-N 273 demonstrated around 30% of full collagenolytic activity regardless of the heterozygous or homozygous state. 3D-molecular envelope modelling using small angle X-ray scattering demonstrated altered positioning of the catalytic domain within dimeric molecules. Taken together, our data suggest that rs1042704 directly contributes to the fibrotic phenotype of DD.

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  1. eLife

    ###Reviewer #3:

    The paper from Itoh is a thorough and interesting analysis of a mechanistic dissection of the underlying cause of Dupuytren's Disease (DD). One exonic SNP is associated with the disease and this mutation changes a residue in helix C of MMP14, a major collagenase, from Asp to Asn. Interestingly, helix C is distant to the catalytic center and the authors show not unexpectedly that recombinant mutant forms of the protease bearing the mutation have identical gelatinolytic and collagenolytic activity in solution. However, in the cell membrane bound form, collagenolysis is markedly reduced. The authors discuss several possibilities for this centering on the potential impaired ability to form dimers. Dimerization and collagen binding has been shown by many groups (please cite some other groups and not just your labs work) to be important for collagen triple helicase activity. This is then suggested to be the underlying cause of the defect in collagenolysis (that then leads to impaired collagen turnover and hence the build up of collagen at several locations in these patients with DD).

    As always there are several points that need addressing to make this a truly nice piece of analysis and data. The major criticism resides in the very nice patient data presented in figure 5. This is key to the whole paper but sadly the authors actually ignore what is shown and drive forward with their own interpretation of the underlying mechanism.

    Major comments:

    1. It is quite clear from a variety of approaches used in the detailed analyses in Fig 5 that there is a strong difference in the degree of enzyme activation occurring in the patient and normal cells comparing AA, which shows the predominant fully active ~51k form vs GG very low amounts perhaps 5% of the mutant when on the cell surface. (the gels are poor quality and so the estimate of MW is difficult to be sure). Thus, the simplest explanation for the reduced collagenolytic activity of the patient is that there is less active protease, without invoking alternate mechanisms. Nonetheless, I understand why the authors investigated dimerization and hemopexin domain interactions and that is fair enough. BUT, those data and interpretations need to be placed in context with fig 5. The interpretation is that other effects occur that alter the activation of MMP14 buy furin or in its cell surface protein protein interactions or with the plasma membrane

    2. Relatively few analyses have been performed of the critical residues in collagenases for collagenolysis. In MMP8 re the S3' site reveals the importance of specific residues in contacting collagen for cleavage (Pelman) that apparently is not important for the mutation under study in the present paper as 237 is distant from the active site on Helix C. Notably, 237 lies in an interesting sequence: DDDRR in which one of the Asp couples to the active site in triple salt bridge relay commencing from the NH2 of the F/Y at the start of the catalytic domain after correct activation, and this is needed to fully activate MMPs. This work by Stoecker should be referenced (though it is not in relation to MMP14 it is a general principle for all MMPs). Please discuss this D as it may affect the electrostatic environment of the 273 position and so reduce catalytic potential. While evidence presented does not indicate this (for collagen and gelatin) there are no kcat/km determinations which are needed to quantify the effect of the mutation.

    3. However, the 273 position is potentially close to the top (blade I) of the adjacent hemopexin domain that the authors know very well is key for collagenolytic activity. The authors posit quite correctly that the mutation may affect the interaction with the hemopexin domain and I totally agree. Collagenolytic activity is difficult and precision in protein contacts is likely needed for catalysis to occur. A model of the catalytic domain contacting the hemopexin domain in blade I is needed to help interpret this. See Zhao et al 2014 (http://dx.doi.org/10.1016/j.str.2014.11.021 ). With the Xray scattering data this appears to be a potential mechanism for disruption, not just dimerization. Please include in Fig 1 a model of the full length MT1-MMP and the site of 273 in relation to the top B strand of blade I for the potential interaction by modelling. Arg 360 by eye might be a potential interactor. Though there are two other Arg that may be involved perhaps R 330, R343 and R345? Please investigate this as it will be interesting.

    4. In this regard, a major oversight has been the lack of reference to the very good analyses of MT1-MMP membrane association by Marcinket al (2019) Structure 27: 281-292.e6. This reveals the membrane binding associations of blade III and IV of the Hx domain which differentially orients the protease on the surface and hence to collagen. An earlier paper by the same group (http://dx.doi.org/10.1016/j.str.2014.11.021 ) also has been ignored (above). These analyses are extremely detailed with amino acid resolution and much could be gained by interpreting these contact residues between collagen and the hemopexin domain and the domain and lipids and hence how it interacts with the catalytic domain where the mutation resides. This must be done in depth to be fair to other work and also for deeper biological insight to the mechanism of collagenolysis in general and in these patients in particular. The membrane association may also drive or supplement dimerization.

    5. I have a serious issue with the fusion construct used in Fig 6. "The Fc part of these chimera molecules enforces the ectodomain of the enzymes to form a disulfide bonds-mediated stable homodimer (Figure 6B), thus allowing the determination of the molecular shape of the MT1-MMP homodimer". How can the authors conclude this? A dimer certainly is formed but its orientation may be totally different from the natural situation where no SS bridge occurs and potentially is in a different orientation. This is a serious caveat that must be clarified to interpret the nice data otherwise in Fig 6.

    6. Only indirect evidence presented that the mutation does not affect dimerization. Please show gel filtration of the complexes or other means to clarify the dimer vs monomeric forms of the WT, mutant and 1/1 heterodimers as this is an obvious and important likely mechanism to explain the phenotype.

    7. It is amazing that the allelic frequency is 0.20. So why does the heterozygous phenotype that the authors investigate in the recombinant experiments show up more in the population?

  2. eLife

    ###Reviewer #2:

    The work contains interesting features, but several aspects of the work are more perplexing than insightful. The authors identify a SNP in MMP14 that occurs in 30% of the population that negatively affects the collagenolytic activity of the encoded gene product, i.e., MT1-MMP. They then propose that the resulting D-to-N mutation may play a role in the pathogenesis of Dupuytren's disease (DD). First, while the title states the the SNP variant causes " .. a defect in collagenolytic activity (that) confers the fibrotic phenotype of DD" , the findings are more appropriately described as having established a correlation between defects in collagenolytic activity and the fibrotic phenotype of DD. However, no data have been presented that document a defect in collagenolytic activity in DD pts harboring the SNP. Indeed, it remains unclear as to whether type I collagen is the key substrate in DD. Given that MT1-MMP can hydrolyze an almost bewildering array of non-collagenous substrates (both cell-surface, secreted and plasma-derived), it is difficult to rule out the possibility that that the D-to-N mutation does not more profoundly affect the hydrolysis of an alternate target. It would be interesting to know if there are changes in gene expression when COS cells are transfected with wt vs the SNP variant of MT1-MMP and cultured on plastic (or even with an E-to-A mutation in the catalytic domain). Second, these concerns notwithstanding, if one were to assume that type I collagen is the critical target, the underlying mechanisms that impact collagenolytic activity are unclear. The authors document complex changes in MT1-MMP processing and cell surface expression in combination with structural changes in the soluble homodimer. Yet, when the soluble variant was shown to express normal type I collagenolytic activity, a conclusion was reached that enzyme activity is likely affected "only when the proteinase is expressed on the cell surface." Possibly, but how do we rule out effects on MT1-MMP exocytosis, endocytosis,trafficking or post-translational modifications in the tail, hinge region, etc - or as mentioned above, hydrolysis of an alternate - and potentially more important - target?

  3. eLife

    ###Reviewer #1:

    In this paper the authors focus on a mutation of MT1-MMP that seems to be associated with Dupuytren's Disease (DD). Using overexpression systems and cells isolated from patients they provide evidence that a major defect of the mutant form of MT1-MMP is it’s reduced ability to activate MMP-2 activation and in turn collagen degradation. Although interesting, the paper presents major shortcomings.

    -All the results obtained are based on in vitro experiments and most of the studies are dependent on overexpression systems.

    -The effects of mutant MT1-MMP on MMP2 activation are not as impressive as the authors claim. No statistical analysis is provided for Fig. 2B (MMP2 activation in cells expressing WT or mutated form of MT1-MMP) and it is not clear if the changes in MMP2 activation observed in Figure 3B (pro-MMP2 activation in cells from patients) are indeed significant. From the graph presented it does not seem to be the case. If this is the case, then the major point of the paper is indeed not corroborated by strong evidence.

    -The authors propose that WT and mutated MT-MMP might form a dimer and the mutated form might act as dominant negative. IP is shown only with anti-FLAG antibodies. Reciprocal IP with anti-myc should also be shown. Also different stringency conditions should be employed to determine the 'strength' of this potential heterodimerization. Importantly advanced FRET-based techniques should be used to study and evaluate heterodimers in the plasma membrane.

    -The title of the paper is misleading as these only in vitro based studies do not allow the authors to conclude that the An SNP variant MT1-MMP with a defect in its collagenolytic activity confers the fibrotic phenotype of Dupuytren's Disease. To answer this key question a vertebrate animal model needs to be provided.

    -Figure 5 needs better controls and/or quantification. The IF provided is not convincing and the authors need to provide loading controls of 'surface' proteins. Importantly statistical analysis needs to be provided to determine whether the changes observed are significant and important.

    In conclusion it is felt that the major conclusions of this paper are not based on convincing data and more analysis needs to be done in order to determine how exactly the mutated form of MT1-MMP might lead to DD.

  4. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary:

    Although the reviewers recognize that the paper contains interesting features, they also addressed major concerns and pitfalls with the study, including: 1) the overall significance; 2) lack of in depth mechanism whereby MT1-MMP variants might alter collagenolytic activity; 3) lack of functional studies with cells isolated from DD patients; 4) the importance of type I collagen as a key substrate in DD remains unclear; and 6) lack of solid evidence that MT1-MMP itself plays a key role in DD.

  5. Version published to 10.1101/2020.06.09.142513 on bioRxiv