High-temperature Requirement Protein A1 Research Articles

High-Temperature Requirement Protein A1 (HtrA1) Gene Expression in Dog Mandible Bone by <I>β</I>-TCP

Beta-tricalcium phosphate (β-TCP) has been used for bone regeneration in a variety of surgical procedures including dental implant therapy with satisfactory clinical results. However, very little is known about the molecular basis mechanisms enhancing the bone formation by β-TCP. To understand the molecular basis mechanism of β-TCP in bone formation, β-TCP was implanted into bone defects of mandible bones in beagle dogs. After 4, 7 and 14 days, bone tissues during the healing process were recovered and gene expression profiles were examined using an Affymetrix GeneChip system. A significantly higher high-temperature requirement protein A1 (HtrA1) mRNA level was found in the 4-day samples after β-TCP implantation compared with the controls. The elevated HtrA1 gene expressions in β-TCP-implanted bone tissues were confirmed by RT-PCR and real-time PCR. Because HtrA1 is known as a key regulator of physiological and pathological matrix mineralization, the enhancement of HtrA1 gene expression in β-TCP-implanted mandible bone might be one of the molecular mechanisms for stimulating bone formation.

HtrA1: a novel regulator of physiological and pathological matrix mineralization?

HtrA1 (high-temperature requirement protein A1) is a secreted multidomain protein with proven serine protease activity and the ability to regulate TGF-beta (transforming growth factor-beta)/BMP (bone morphogenetic protein) signalling. There is increasing evidence that HtrA1 regulates several pathological processes, including tumour development, Alzheimer's disease, age-related macular degeneration and osteoarthritis, although the mechanism(s) by which it regulates these processes have not been fully elucidated. Using overexpression and knock-down strategies, we have evidence demonstrating that HtrA1 is also a key regulator of physiological and pathological matrix mineralization in vitro. We propose that HtrA1 regulates mineralization by inhibiting TGF-beta/BMP signalling and/or by cleaving specific matrix proteins, including decorin and MGP (matrix Gla protein). Taken together, these studies suggest that HtrA1 may be a novel therapeutic target for several diseases.

E. coli에서 발현된 human HtrA1 단백질의 정제와 HtrA1의 serine protease 활성 조건에 관한 연구

E. coli HtrA (High temperature requirement protein A)의 human homologue 중 하나인 HtrA1은 IGFBP를 절단하여 IGF의 활동을 조절하는 serine protease으로 알려졌다. HtrA1의 serine protease 활성이 여러 질병의 발병 mechanism과 연관성을 가진 것으로 예상되고 있지만, 이런 상관관계를 밝히기 위해서 기본적으로 필요한 다량의 HtrA1 단백질의 발현 및 정제조건과 HtrA1 serine protease의 최적 활성조건이 확립되어 있지 않은 상황이다. 따라서 본 연구에서는 pGEX 시스템을 이용하여 E. coli에서 mature HtrA1인 <TEX>${\Delta}149(WT)$</TEX>와 catalytic site mutant인 <TEX>${\Delta}149(S328A)$</TEX>를 85%의 순도로 1 liter 배양 시, 정제된 단백질을 각각 <TEX>$400{\mu}g,\;520{\mu}g$</TEX> 얻을 수 있는 발현조건을 정립하였다. 또한 HtrA1 serine protease 활성은 protease의 농도와 substrate와의 반응시간에 dependent하며, substrate와의 반응온도가 <TEX>$42^{\circ}C$</TEX>일 때 최적의 serine protease활성을 나타내는 것을 알 수 있었다. 특히 <TEX>$200{\mu}M$</TEX>의 HtrA1 serine protease를 <TEX>$37^{\circ}C$</TEX>에서 3시간 반응 시켰을 때, substrate로 사용한 <TEX>${\beta}-casein$</TEX>의 약 50%가 절단되는 것을 관찰하였다. 따라서 이 반응조건에 사용한 HtrA1의 양을 1 unit으로 하여 HtrA1의 serine protease활성을 여러 조건에서 비교 분석할 수 있다 본 연구에서 정립한 mature HtrA1을 다량으로 얻을 수 있는 발헌 및 정제조건과 serine protease 최적 활성조건은 HtrA1의 serine protease 활성과 생물학적 기능의 상관관계를 이해하는데 활용될 수 있을 것이다. Human HtrA1 (High temperature requirement protein A1) is a homologue of the E. coli periplasmic serine protease HtrA. A recent study has demonstrated that HtrA1 is a serine protease involved in processing of insulin like growth factor binding protein (ICFBP), indicating that it serves as an important regulator of IGF activity. Additionally, several lines of evidence suggest a striking correlation between proteolytic activity of HtrA1 serine protease and the pathogenesis of several diseases; however, physiological roles of HtrA1 remain to be elucidated. We used the pGEX bacterial expression system to develop a simple and rapid method for purifying HtrA1, and the recombinant HtrA1 protein was utilized to investigate the optimal conditions in executing its proteolytic activity. The proteolytically active HtrA1 was purified to approximately 85% purity, although the yield of the recombinant HtrA1 protein was slightly low <TEX>$460{\mu}g$</TEX> for 1 liter E. coli culture). Using in vitro endoproteolytic cleavage assay, we identified that the HtrA1 serine protease activity was dependent on the enzyme concentration and the incubation time and that the best reaction temperature was <TEX>$42^{\circ}C$</TEX> instead of <TEX>$37^{\circ}C$</TEX>. We arbitrary defined one unit of proteolytic activity of the HtrA1 serine protease as 200nM of HtrA1 that cleaves half of <TEX>$5{\mu}M\;of\;{\beta}-casein$</TEX> during 3 hr incubation at <TEX>$37^{\circ}C$</TEX>. Our study provides a method for generating useful reagents to investigate the molecular mechanisms by which HtrA1 serine protease activity contributes in regulating its physiological function and to identify natural substrates of HtrA1.