On-column activation of bovine recombinant metalloproteinase 20

Wang, Shengwu; Zhu, Judy; Zhang, Yan; Li, Ling; DenBesten, Pamela; Wu, Li

doi:10.1016/j.ab.2005.11.045

Cited by 4 publications

(5 citation statements)

References 13 publications

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“…Recombinant human amelogenin (rh174) and MMP20 were produced and purified as previously described (Li et al, 1999; Wang et al, 2006). Commercial recombinant human KLK4 was purchased from R&D Systems (Minneapolis, MN, USA).…”

Section: Methodsmentioning

confidence: 99%

Preferential and selective degradation and removal of amelogenin adsorbed on hydroxyapatites by MMP20 and KLK4 in vitro

et al. 2014

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The hardest tooth enamel tissue develops from a soft layer of protein-rich matrix, predominated by amelogenin that is secreted by epithelial ameloblasts in the secretory stage of tooth enamel development. During enamel formation, a well-controlled progressive removal of matrix proteins by resident proteases, Matrix metalloproteinase 20 (MMP20), and kallikrein 4 (KLK4), will provide space for the apatite crystals to grow. To better understand the role of amelogenin degradation in enamel biomineralization, the present study was conducted to investigate how the adsorption of amelogenin to hydroxyapatite (HAP) crystals affects its degradation by enamel proteinases, MMP20 and KLK4. Equal quantities of amelogenins confirmed by protein assays before digestions, either adsorbed to HAP or in solution, were incubated with MMP20 or KLK4. The digested samples collected at different time points were analyzed by spectrophotometry, SDS-PAGE, high performance liquid chromatography (HPLC), and LC-MALDI MS/MS. We found that majority of amelogenin adsorbed on HAP was released into the surrounding solution by enzymatic processing (88% for MMP20 and 98% for KLK4). The results show that as compared with amelogenin in solution, the HAP-bound amelogenin was hydrolyzed by both MMP20 and KLK4 at significantly higher rates. Using LC-MALDI MS/MS, more accessible cleavage sites and hydrolytic fragments from MMP20/KLK4 digestion were identified for the amelogenin adsorbed on HAP crystals as compared to the amelogenin in solution. These results suggest that the adsorption of amelogenin to HAP results in their preferential and selective degradation and removal from HAP by MMP20 and KLK4 in vitro. Based on these findings, a new degradation model related to enamel crystal growth is proposed.

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Section: Methodsmentioning

confidence: 99%

Preferential and selective degradation and removal of amelogenin adsorbed on hydroxyapatites by MMP20 and KLK4 in vitro

et al. 2014

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“…KEY WORDS: amelogenin, matrix metalloproteinase 20, protein interaction, tooth enamel, biomineralization, amelogenesis imperfecta. expression and purification, and when the enzyme was being used to degrade its substrates (Li et al, 1999;Wang et al, 2006). The results of this study provide new insights into the pathogenesis of AI, and are also helpful in our understanding of the interactions between amelogenin and MMP20 during tooth enamel formation.…”

Section: Introductionmentioning

confidence: 84%

“…Both rhMMP20 and E227A inactive rhMMP20 were characterized by SDS-PAGE and Western blot analysis. Enzymatic activities of the purified proteases were analyzed by zymography and quenched-peptide assay (Li et al, 1999;Wang et al, 2006). The digested amelogenins were visualized by SDS-PAGE and analyzed by MALDI-TOF mass spectrometry (MS).…”

Section: Methodsmentioning

confidence: 99%

“…In this study, we used different methods to analyze the amelogenin-MMP20 interactions, including a state-of-theart technique, surface plasmon resonance (SPR), for our quantitative and dynamic experiments. For these protein-protein interaction assays, we used an intact and inactive MMP20 protein, which was created by introducing a point mutation, Glu225 to Ala, in its catalytic domain, to prevent interference from the smaller fragments produced by autolysis of this protein during its expression and purification, and when the enzyme was being used to degrade its substrates (Li et al, 1999;Wang et al, 2006). The results of this study provide new insights into the pathogenesis of AI, and are also helpful in our understanding of the interactions between amelogenin and MMP20 during tooth enamel formation.…”

Section: Introductionmentioning

confidence: 99%

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Reduced Amelogenin-MMP20 Interactions in Amelogenesis Imperfecta

Tanimoto

Zhu

et al. 2008

J Dent Res

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Amelogenin with a proline 41 to threonine mutation (P41T) is hydrolyzed at a lower rate by matrix metalloproteinase 20 (MMP20), resulting in an inherited tooth enamel defect, amelogenesis imperfecta (AI). The aim of this study was to elucidate the effect of P41T on the interactions between amelogenin and MMP20, which may contribute to the formation of this type of AI. The interactions of a recombinant wild-type human amelogenin and its P41T mutant with recombinant human MMP20 were compared by substrate competition assay, pull-down assay, and surface plasmon resonance (SPR). The results showed that the binding of the P41T mutant amelogenin for MMP20 was significantly lower than that of wild-type amelogenin. Our study supports a model in which the P41T mutation reduces the interactions between amelogenin and MMP20, leading to decreased degradation of amelogenin by MMP20, and resulting in AI.

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“…Although KLK4 activity is calcium-independent, calcium may still indirectly affect KLK4 activity in the Nckx4 −/− enamel matrix through MMP20. MMP20 is a well-known calcium-dependent metalloproteinase, which can activate KLK4 in vitro ( Wang et al, 2006 ; Hadler-Olsen et al, 2011 ; Yamakoshi et al, 2013 ). Thus, the efficiency of MMP20 remaining in the early maturation stage of the enamel matrix may be adversely affected by the reduced calcium, subsequently decreasing the activity of KLK4 in the maturation stage of Nckx4 −/− enamel matrix.…”

Section: Discussionmentioning

confidence: 99%

Sodium/(calcium + potassium) exchanger NCKX4 optimizes KLK4 activity in the enamel matrix microenvironment to regulate ECM modeling

Chan

Cheng²,

Jalali³

et al. 2023

Front. Physiol.

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Enamel development is a process in which extracellular matrix models from a soft proteinaceous matrix to the most mineralized tissue in vertebrates. Patients with mutant NCKX4, a gene encoding a K+-dependent Na+/Ca2+—exchanger, develop a hypomineralized and hypomature enamel. How NCKX4 regulates enamel protein removal to achieve an almost protein-free enamel is unknown. We characterized the upregulation pattern of Nckx4 in the progressively differentiating enamel-forming ameloblasts by qPCR, and as well as confirmed NCKX4 protein to primarily localize at the apical surface of wild-type ruffle-ended maturation ameloblasts by immunostaining of the continuously growing mouse incisors, posing the entire developmental trajectory of enamel. In contrast to the normal mature enamel, where ECM proteins are hydrolyzed and removed, we found significant protein retention in the maturation stage of Nckx4−/− mouse enamel. The Nckx4−/− enamel held less Ca2+ and K+ but more Na+ than the Nckx4+/+ enamel did, as measured by EDX. The alternating acidic and neutral pH zones at the surface of mineralizing Nckx4+/+ enamel were replaced by a largely neutral pH matrix in the Nckx4−/− enamel. In situ zymography revealed a reduced kallikrein-related peptidase 4 (KLK4) activity in the Nckx4−/− enamel. We showed that KLK4 took on 90% of proteinase activity in the maturation stage of normal enamel, and that recombinant KLK4 as well as native mouse enamel KLK4 both performed less effectively in a buffer with increased [Na+] and pH, conditions found in the Nckx4−/− developing enamel. This study, for the first time to our knowledge, provides evidence demonstrating the impaired in situ KLK4 activity in Nckx4−/− enamel and suggests a novel function of NCKX4 in facilitating KLK4-mediated hydrolysis and removal of ECM proteins, warranting the completion of enamel matrix modeling.

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On-column activation of bovine recombinant metalloproteinase 20

Cited by 4 publications

References 13 publications

Preferential and selective degradation and removal of amelogenin adsorbed on hydroxyapatites by MMP20 and KLK4 in vitro

Preferential and selective degradation and removal of amelogenin adsorbed on hydroxyapatites by MMP20 and KLK4 in vitro

Reduced Amelogenin-MMP20 Interactions in Amelogenesis Imperfecta

Sodium/(calcium + potassium) exchanger NCKX4 optimizes KLK4 activity in the enamel matrix microenvironment to regulate ECM modeling

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