Zanetti M, Litteri L, Griffiths G, Gennaro R, Romeo D
Dipartimento di Biochimica, Università di Trieste, Italy.
J Immunol. 1991 Jun 15;146(12):4295-300.
The antimicrobial polypeptides Bac7 and Bac5 (bactenecins) are stored in the large granules of bovine neutrophils as precursor forms, or probactenecins. Maturation of probactenecins has been investigated by studying the effects of stimulus-induced degranulation on this process. Stimulation of neutrophils with PMA, which is a secretagogue for specific and large granules but not for azurophils, induces a substantial discharge of uncleaved probactenecins in the extracellular medium, as revealed by Western blot analysis. When neutrophils are exposed to opsonized bacteria in the presence of cytochalasin B, resulting in exocytosis of the content of azurophils in addition to that of specific and large granules, probactenecins are secreted and rapidly converted into the corresponding mature antimicrobial peptides. Such a conversion is prevented if serine proteases, stored in the azurophils, are inhibited by pretreatment of neutrophils with PMSF. Phagocytosis, while causing a rapid discharge of the contents of azurophil and of the large granules into phagocytic vacuoles, as indicated by immunogold electron microscopy, also induces cleavage of probactenecins into mature peptides, as revealed by Western blot analysis. We conclude that the final processing of the storage forms of bactenecins arises from their interaction with the serine protease(s) of azurophils during bacteria-induced degranulation of neutrophils.
抗菌多肽Bac7和Bac5（杆菌防御素）以前体形式，即原杆菌防御素，储存在牛嗜中性粒细胞的大颗粒中。通过研究刺激诱导的脱颗粒对这一过程的影响，对原杆菌防御素的成熟进行了研究。用佛波酯刺激嗜中性粒细胞，佛波酯是特异性和大颗粒而非嗜天青颗粒的促分泌剂，Western印迹分析显示，它会诱导大量未切割的原杆菌防御素释放到细胞外培养基中。当中性粒细胞在细胞松弛素B存在下暴露于调理过的细菌时，除了特异性和大颗粒的内容物外，还会导致嗜天青颗粒的内容物胞吐，原杆菌防御素会被分泌并迅速转化为相应的成熟抗菌肽。如果用苯甲基磺酰氟预处理中性粒细胞来抑制储存在嗜天青颗粒中的丝氨酸蛋白酶，这种转化就会被阻止。吞噬作用虽然会导致嗜天青颗粒和大颗粒的内容物迅速释放到吞噬泡中，免疫金电子显微镜显示了这一点，Western印迹分析也显示，它还会诱导原杆菌防御素切割成成熟肽。我们得出结论，杆菌防御素储存形式的最终加工源于它们在细菌诱导的中性粒细胞脱颗粒过程中与嗜天青颗粒的丝氨酸蛋白酶相互作用。
