Abstract
Adipocytes attached to the extracellular matrix (ECM) mainly consist of collagen in adipose tissues, while the degradation of ECM by collagenase induces the apoptosis of adipocytes, leading to a decrease in local subcutaneous adipose. To achieve this goal, we are developing a mutant collagenase H (ColH) to remove local subcutaneous fat such as submental fat (SMF). Three vectors were constructed for expressing rColH(FM, mutant for fat melting, with 6xHis tag), rColH(WT, wild-type, with 6xHis tag), and rColH(E451D, E451D mutant, without 6xHis tag) in Escherichia coli. rColH(FM) & rColH(WT) were purified by Ni Sepharose on a laboratory scale, while rColH(E451D) was purified by five chromatography purification steps on a large scale. Then, the stability of rColH(FM) and rColH(WT) was tested by SDS-PAGE to investigate the influence of the E451D mutation on stability. Afterwards, the enzyme kinetics of ColH (mutant or wild-type, with or without His tag) were investigated and compared. Finally, the adipolysis of rColH(E451D) at various doses was tested in vitro and in vivo. The ultrasound results in minipigs suggested that effective adipolysis was induced by rColH(E451D) compared with the negative control, and the histological results suggest dose-dependent fibrosis, necrosis, inflammation and cholesterol cleft formation. These findings indicate the possibility of rColH(E451D) becoming a new injectable drug to safely remove subcutaneous adipose.
Highlights
Collagenase from Clostridium histolyticum was first isolated and reported in 1953 [1]
After storage at 4 ̊C or RT for 8 weeks, no autolysis was observed with SDS-PAGE for rColH(WT) or rColH(FM) (Fig 1C), and their enzyme activities were stable at 4 ̊C but decreased at RT (Fig 1D)
The results indicated that the E451D mutation did not influence the stability of the enzyme but resulted in lower specific activity because E451 is the key position in the catalytic domain [22]
Summary
Collagenase from Clostridium histolyticum was first isolated and reported in 1953 [1]. Compared with Type I collagenases, collagenase H (z subtype), a Type II collagenase, has increased activity on synthetic peptide substrates. These two types of collagenases are both metalloproteinases that can digest collagen, the main component of the ECM in animal tissues [5]. Due to the specific collagenolytic properties of collagenase from C. histolyticum, clostridial collagenase was used to treat tissues from patients with Peyronie’s disease [6] and was injected into Dupuytren’s cords from patients undergoing fasciotomy [7].
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