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. 2020 Mar 3;10(1):3927.
doi: 10.1038/s41598-020-60802-z.

Recombinant Collagenase From Grimontia Hollisae as a Tissue Dissociation Enzyme for Isolating Primary Cells

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Free PMC article

Recombinant Collagenase From Grimontia Hollisae as a Tissue Dissociation Enzyme for Isolating Primary Cells

Keisuke Tanaka et al. Sci Rep. .
Free PMC article

Abstract

Collagenase products are crucial to isolate primary cells in basic research and clinical therapies, where their stability in collagenolytic activity is required. However, currently standard collagenase products from Clostridium histolyticum lack such stability. Previously, we produced a recombinant 74-kDa collagenase from Grimontia hollisae, which spontaneously became truncated to ~60 kDa and possessed no stability. In this study, to generate G. hollisae collagenase useful as a collagenase product, we designed recombinant 62-kDa collagenase consisting only of the catalytic domain, which exhibits high production efficiency. We demonstrated that this recombinant collagenase is stable and active under physiological conditions. Moreover, it possesses higher specific activity against collagen and cleaves a wider variety of collagens than a standard collagenase product from C. histolyticum. Furthermore, it dissociated murine pancreata by digesting the collagens within the pancreata in a dose-dependent manner, and this dissociation facilitated isolation of pancreatic islets with masses and numbers comparable to those isolated using the standard collagenase from C. histolyticum. Implantation of these isolated islets into five diabetic mice led to normalisation of the blood glucose concentrations of all the recipients. These findings suggest that recombinant 62-kDa collagenase from G. hollisae can be used as a collagenase product to isolate primary cells.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Graphical representation of recombinant Grimontia hollisae collagenases and their collagenolytic activities. (A) Recombinant proteins were designed as 74 kDa (aa 88–767, including the PPC domain), 62 kDa (aa 88–646), and 60 kDa (aa 88–624) of G. hollisae collagenase. The amino acid sequence of the linker region is highlighted and four G-X-Y repeats are underlined. (B) Recombinant collagenases were purified from Brevibacillus culture medium by diethylaminoethanol-Sepharose chromatography. Ten microliters of culture media (left panel) and two micrograms of purified recombinant collagenases (right panel) were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 7.5% polyacrylamide gel. Numbers on the left are molecular masses (in kDa) of the markers. (C) Collagenolytic activities of recombinant collagenases were determined using FITC-collagen. Values represent the average of triplicate trials ± standard deviation. *P < 0.01, determined by one-way analysis of variance. The uncropped gel is included in a Supplementary Information File. (D) Stability analysis of recombinant 74-kDa collagenase. Recombinant collagenase was incubated at 37 °C in 50 mM Bis-Tris-HCl buffer (pH 7.5) containing 0.2 M NaCl and 5 mM CaCl2. After incubation for various time intervals, the reaction mixture was analysed by SDS-PAGE (left panel) and its collagenolytic activity was measured using FITC-labelled type I collagen (right panel). Values represent the average of triplicate trials ± standard deviation. (E) Collagenolytic activities of recombinant collagenases with different proportions of 74-kDa and 62-kDa proteins were determined using FITC-collagen. Values represent the average of triplicate trials ± standard deviation. The uncropped gel is included in a Supplementary Information File.
Figure 2
Figure 2
Characterisation of recombinant 62-kDa collagenase from Grimontia hollisae. (A) Purified recombinant 62-kDa collagenase was analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (lane 1) and real-time gelatin zymography (lane 2). Numbers on the left are molecular masses (in kDa) of the markers. (B) Size exclusion chromatogram of recombinant 62-kDa collagenase. Size exclusion chromatography was performed on an Alliance 2895 system using a Superdex 200 HR10/30 column. The sample was loaded onto a column and eluted in an isocratic manner with 50 mM Bis-Tris-HCl (pH 7.5) containing 0.2 M NaCl, at a flow rate of 0.75 ml/minute. The separated protein fraction was detected at 220 nm. (C) pH-dependence of recombinant 62-kDa collagenase. Collagenase and FALGPA were mixed in each buffer mentioned below, then incubated at 30 °C for 5 minutes. The following buffers were used: 50 mM MES (pH 6.0–7.0), 50 mM HEPES (pH 7.0–8.5), 50 mM TAPS (pH 8.5 and 9.0), and 50 mM CHES (pH 9.0 and 10.0) containing 0.2 M NaCl and 5 mM CaCl2. (D) Temperature-dependence of recombinant 62-kDa collagenase. Collagenase was incubated with the Pz peptide in 50 mM HEPES (pH 7.5) containing 0.2 M NaCl and 5 mM CaCl2 at various temperatures (10–60 °C). (E) Stability analysis of recombinant 62-kDa collagenase. Recombinant collagenase was incubated at 37 °C in 50 mM Bis-Tris-HCl buffer (pH 7.5) containing 0.2 M NaCl and 5 mM CaCl2. After incubation for various time intervals, the reaction mixture was analysed by SDS-PAGE (left panel) and its collagenolytic activity was measured using FITC-labelled type I collagen (right panel). Values represent the average of triplicate trials ± standard deviation.
Figure 3
Figure 3
Collagen cleavage assay. Purified recombinant 62-kDa collagenase from Grimontia hollisae was incubated with type I (A), type II (B), type III (C), type IV (D), type V (E), and type VI (F) collagens at 30 °C. After the reaction was stopped by the addition of 1/4 volume of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer at each time point, each sample was analysed by SDS-PAGE using a 7.5% polyacrylamide gel. The uncropped gel is included in a Supplementary Information File.
Figure 4
Figure 4
Quantification of collagen and tissue weights in the three fractions after pancreas dissociation. (A) Graphical representation of experimental procedure. (B) Undissociated, soluble dissociated, and insoluble dissociated fractions were prepared after pancreas dissociation using recombinant Grimontia hollisae collagenase (solid line) or purified Clostridium histolyticum collagenase (dashed line) with thermolysin (0.012 mg/ml). All fractions from collagenase-treated pancreata were hydrolysed by heating at 110 °C with 6 M HCl for 20 hours. Quantification of hydroxyproline content was performed using a 3200 QTRAP mass spectrometer; the collagen weight in each fraction was converted from hydroxyproline values. The total amino acid content of each fraction was measured with a L-8800 amino acid analyser; the tissue weight of each fraction was converted from the total amino acid content.
Figure 5
Figure 5
Determination of isolated islet number and islet equivalent. Pancreas dissociation was performed using recombinant Grimontia hollisae collagenase (solid line) or purified Clostridium histolyticum collagenase (dashed line) with thermolysin (0.012 mg/ml). Islets were purified from insoluble dissociated fractions using density-gradient centrifugation and stained with dithizone. Islet number (A) and islet equivalent (B) were determined based upon photos of all isolated islets in each fraction, which were taken using a charge-coupled device digital camera.
Figure 6
Figure 6
Transplantation of primary islet cells into diabetic mice. (A) Optical image of primary islet cells (left panel) and the transplantation of 300 islet cells into the subrenal capsular space of a recipient mouse (right panel). Scale bars, 500 μm (left); 2 mm (right). (B) Change in blood glucose concentrations of five diabetic mice that received 300 islet cells (solid lines) and three diabetic mice that did not undergo transplantation (dashed lines). Nephrectomy of the graft-bearing kidney was performed at 38 days after transplantation. (C) Intraperitoneal glucose tolerance tests of four diabetic mice that received 300 islet cells (solid lines), three diabetic mice that did not undergo transplantation (coloured dashed lines), and five healthy mice (black dashed lines) at 34 days after transplantation. (D) Histopathological analysis of sections of resected left kidney bearing transplanted islets in the subrenal capsule. Haematoxylin and eosin (H&E) staining (left panel) and immunohistochemical anti-insulin staining were performed. Scale bar, 100 μm.

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