Department of Microbiology, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Krakow 30-387, Poland.
Biol Chem. 2010 Jan;391(1):105-17. doi: 10.1515/BC.2010.009.
Proteases of Tannerella forsythia, a pathogen associated with periodontal disease, are implicated as virulence factors. Here, we characterized a matrix metalloprotease (MMP)-like enzyme of T. forsythia referred to as karilysin. Full-length (without a signal peptide) recombinant karilysin (49.9 kDa) processed itself into the mature 18-kDa enzyme through sequential autoproteolytic cleavage at both N- and C-terminal profragments. The first cleavage at the Asn14-Tyr15 peptide bond generated the fully active enzyme (47.9 kDa) and subsequent truncations at the C-terminus did not affect proteolytic activity. Mutation of Tyr15 to Ala generated a prokarilysin variant that processed itself into the final 18-kDa form with greatly reduced kinetics. Inactive prokarilysin with the mutated catalytic Glu residue (E136A) was processed by active karilysin at the same sites as the active enzymes. Karilysin proteolytic activity and autoprocessing were inhibited by 1,10-phenanthroline and EDTA. Calcium ions were found to be important for both the activity and thermal stability of karilysin. Using CLiPS technology, the specificity of karilysin was found to be similar to that of MMPs with preference for Leu/Tyr/Met at P1' and Pro/Ala at P3. This specificity and the ability to degrade elastin, fibrinogen and fibronectin may contribute to the pathogenicity of periodontitis.
福赛拟杆菌蛋白酶与牙周病有关，被认为是致病因子。在这里，我们描述了福赛拟杆菌的一种基质金属蛋白酶（MMP）样酶，称为卡瑞林酶。全长（无信号肽）重组卡瑞林酶（49.9 kDa）通过在 N-和 C-末端前片段处的连续自蛋白水解切割，自身处理成成熟的 18 kDa 酶。在 Asn14-Tyr15 肽键处的第一次切割产生了完全活性的酶（47.9 kDa），并且随后在 C-末端的截断不影响蛋白水解活性。将 Tyr15 突变为 Ala 生成了一种前卡瑞林酶变体，其自身处理成最终的 18 kDa 形式，但动力学大大降低。具有突变催化Glu残基（E136A）的无活性前卡瑞林酶被活性卡瑞林酶在与活性酶相同的位点进行处理。卡瑞林酶的蛋白水解活性和自蛋白水解被 1,10-菲咯啉和 EDTA 抑制。钙离子对卡瑞林酶的活性和热稳定性都很重要。使用 CLiPS 技术，发现卡瑞林酶的特异性类似于 MMP，对 P1'处的 Leu/Tyr/Met 和 P3 处的 Pro/Ala 有偏好。这种特异性和降解弹性蛋白、纤维蛋白原和纤维连接蛋白的能力可能有助于牙周炎的致病性。
