Diamond D L, Kimball J R, Krisanaprakornkit S, Ganz T, Dale B A
Ciphergen Biosystems, Fremont, CA, USA.
J Immunol Methods. 2001 Oct 1;256(1-2):65-76. doi: 10.1016/s0022-1759(01)00442-2.
Human beta-defensins are antimicrobial peptides that may be critical in the innate immune response to infection. hBD1 and hBD2 are expressed in oral epithelial cells and are detected near the surface of oral tissue, consistent with a role in the epithelial protective barrier function. In this report, we examine secretion of beta-defensins in vitro and in biological fluid using ProteinChip(R) Array, surface enhanced laser desorption/ionization (SELDI) technology combined with time-of-flight mass spectrometry. We show that the 47-amino acid form of hBD1 and the 41-amino acid form of hBD2 are the major secreted forms. These forms are both expressed and secreted under conditions anticipated from previous analysis of beta-defensin mRNAs; specifically, hBD1 is detected in culture supernatant from both unstimulated and stimulated cells, and hBD2 is detected only in stimulated cells. Identity of hBD1 and hBD2 was confirmed by immunocapture on the ProteinChip surface. Both peptides are also present in gingival crevicular fluid that accumulates between the tissue and tooth surface, although hBD1 is also found in several smaller forms suggesting extracellular proteolysis. This methodology offers several technical advantages for detection of defensins in biological fluids, including ease and speed of screening, no need for HPLC preliminary processing, and small sample size.
人β-防御素是抗菌肽，在对感染的固有免疫反应中可能起关键作用。hBD1和hBD2在口腔上皮细胞中表达，并在口腔组织表面附近被检测到，这与其在上皮保护屏障功能中的作用一致。在本报告中，我们使用蛋白质芯片阵列、表面增强激光解吸/电离（SELDI）技术结合飞行时间质谱，研究了体外和生物体液中β-防御素的分泌情况。我们发现，47个氨基酸形式的hBD1和41个氨基酸形式的hBD2是主要的分泌形式。这些形式在根据先前对β-防御素mRNA的分析所预期的条件下表达和分泌；具体而言，在未刺激和刺激细胞的培养上清液中均检测到hBD1，而仅在刺激细胞中检测到hBD2。通过在蛋白质芯片表面进行免疫捕获确认了hBD1和hBD2的身份。两种肽也存在于组织和牙齿表面之间积聚的龈沟液中，尽管hBD1也以几种较小的形式存在，提示细胞外蛋白水解。这种方法在检测生物体液中的防御素方面具有几个技术优势，包括筛选简便、速度快、无需HPLC预处理以及样本量小。
