Leo Fredrik, Svensäter Gunnel, Lood Rolf, Wickström Claes
Department of Oral Biology and Pathology, Faculty of Odontology, Malmö University, Malmö, Sweden.
Genovis AB, Lund, Sweden.
Front Microbiol. 2023 Feb 28;14:1127466. doi: 10.3389/fmicb.2023.1127466. eCollection 2023.
MUC5B is the predominant glycoprotein in saliva and is instrumental in the establishment and maintenance of multi-species eubiotic biofilms in the oral cavity. Investigations of the aciduric family, and its role in biofilms emphasizes the diversity across different genera of the proteolytic systems involved in the nutritional utilization of mucins. We have characterized a protease from , MdpL (Mucin degrading protease from ) with a high protein backbone similarity with commensals that exploit mucins for attachment and nutrition. MdpL was shown to be associated with the bacterial cell surface, in close proximity to MUC5B, which was sequentially degraded into low molecular weight fragments. Mapping the substrate preference revealed multiple hydrolytic sites of proteins with a high O-glycan occurrence, although hydrolysis was not dependent on the presence of O-glycans. However, since proteolysis of immunoglobulins was absent, and general protease activity was low, a preference for glycoproteins similar to MUC5B in terms of glycosylation and structure is suggested. MdpL preferentially hydrolyzed C-terminally located hydrophobic residues in peptides larger than 20 amino acids, which hinted at a limited sequence preference. To secure proper enzyme folding and optimal conditions for activity, incorporates a complex system that establishes a reducing environment. The importance of overall reducing conditions was confirmed by the activity boosting effect of the added reducing agents L-cysteine and DTT. High activity was retained in low to neutral pH 5.5-7.0, but the enzyme was completely inhibited in the presence of Zn. Here we have characterized a highly conserved mucin degrading protease from . MdpL, that together with the recently discovered O-glycanase and O-glycoprotease enzyme groups, increases our understanding of mucin degradation and complex biofilm dynamics.
MUC5B是唾液中的主要糖蛋白，对口腔中多种有益生物膜的形成和维持起着重要作用。对嗜酸菌家族及其在生物膜中的作用的研究强调了参与粘蛋白营养利用的不同属蛋白水解系统的多样性。我们从[具体来源]中鉴定出一种蛋白酶MdpL（来自[具体来源]的粘蛋白降解蛋白酶），它与利用粘蛋白进行附着和营养的共生菌具有高度相似的蛋白质骨架。研究表明MdpL与细菌细胞表面相关联，紧邻MUC5B，MUC5B会被依次降解为低分子量片段。对底物偏好性的分析揭示了具有高O-聚糖发生率的蛋白质的多个水解位点，尽管水解并不依赖于O-聚糖的存在。然而，由于不存在免疫球蛋白的蛋白水解作用，且一般蛋白酶活性较低，提示其对糖基化和结构与MUC5B相似的糖蛋白具有偏好性。MdpL优先水解大于20个氨基酸的肽中位于C末端的疏水残基，这暗示了其有限的序列偏好性。为确保酶的正确折叠和最佳活性条件，[具体来源]包含一个建立还原环境的复杂系统。添加的还原剂L-半胱氨酸和二硫苏糖醇的活性增强作用证实了整体还原条件的重要性。在低至中性pH 5.5 - 7.0时仍保留高活性，但在锌存在下酶被完全抑制。在这里，我们鉴定了一种来自[具体来源]的高度保守的粘蛋白降解蛋白酶MdpL，它与最近发现的O-聚糖酶和O-糖蛋白酶酶组一起，增进了我们对粘蛋白降解和复杂生物膜动态的理解。
