Identification of CRAMP, a cathelin-related antimicrobial peptide expressed in the embryonic and adult mouse
Identification of CRAMP, a cathelin-related antimicrobial peptide expressed in the embryonic and adult mouse
复制标题
DOI：
10.1074/jbc.272.20.13088
复制
发表时间：
1997-05-16
影响因子：
4.8
通讯作者：
Gennaro, R
中科院分区：
文献类型：
--
作者：
Gallo, RL;Kim, KJ;Gennaro, R
Cathelicidins是已经在几种哺乳动物物种中鉴定的有效抗微生物肽的前体，先前的工作已经表明该基因家族的成员可以通过其抗微生物作用参与宿主防御并在伤口修复期间激活间充质细胞。为了允许进一步研究这些蛋白质，使用逆转录酶-聚合酶链反应方法来鉴定潜在的小鼠同源物，获得了一个全长562 bp的cDNA克隆，该基因编码一个与其它cathelicidins同源的氨基端prepro结构域和一个独特的羧基端肽，该基因被命名为cathelin相关抗菌肽Cramp，定位于9号染色体上的一个保守的同线性区域，在猪和人中，cathelicidins的基因已经定位于该区域。北方印迹分析检测到一个1-千碱基转录本在成人骨髓和早在E12（血液发育的最早阶段）的胚胎发生过程中表达，逆转录酶-聚合酶链反应还检测到CRAMP在成人睾丸、脾脏、胃和肠中表达，但在脑、肝脏、心脏或骨骼肌中不表达。为了进一步评估CRAMP的表达和功能，合成了对应于预测的COOH-末端区域的肽，CD光谱分析表明CRAMP将形成与其他抗菌肽类似的两亲性α-螺旋，功能研究表明，CRAMP是一种有效的抗生素，通过抑制多种细菌菌株的生长来对抗革兰氏阴性菌（最小抑制浓度0.5-8.0 μ M）和直接以1 μ M透化大肠杆菌的内膜。抗CRAMP的抗血清显示在骨髓前体细胞和中性粒细胞中大量表达，因此，CRAMP代表在小鼠骨髓谱系细胞中发现的第一个抗生素肽。这些数据表明，小鼠中的炎性细胞可以使用非氧化机制杀死微生物，并允许使用小鼠来研究这些肽在宿主防御和伤口修复中发挥的作用。
Cathelicidins are the precursors of potent antimicrobial peptides that have been identified in several mammalian species, Prior work has suggested that members of this gene family can participate in host defense through their antimicrobial effects and activate mesenchymal cells during wound repair, To permit further study of these proteins a reverse transcriptase-polymerase chain reaction approach was used to identify potential mouse homologs, A full-length 562-base pair cDNA clone was obtained encoding an NH2-terminal prepro domain homologous to other cathelicidins and a unique COOH-terminal peptide, This gene, named Cramp for cathelin-related antimicrobial peptide, was mapped to chromosome 9 at a region of conserved synteny to which genes for cathelicidins have been mapped in pig and man. Northern blot analysis detected a 1-kilobase transcript that was expressed in adult bone marrow and during embryogenesis as early as E12, the earliest stage of blood development, Reverse transcriptase-polymerase chain reaction also detected CRAMP expression in adult testis, spleen, stomach, and intestine but not in brain, liver, heart, or skeletal muscle. To evaluate further the expression and function of CRAMP a peptide corresponding to the predicted COOH-terminal region was synthesized, CD spectral analysis showed that CRAMP will form an amphipathic alpha-helix similar to other antimicrobial peptides, Functional studies showed CRAMP to be a potent antibiotic against Gram-negative bacteria by inhibiting growth of a variety of bacterial strains (minimum inhibitory concentrations 0.5-8.0 mu M) and by permeabilizing the inner membrane of Escherichia coli directly at 1 mu M. Antiserum against CRAMP revealed abundant expression in myeloid precursors and neutrophils, Thus, CRAMP represents the first antibiotic peptide found in cells of myeloid lineage in the mouse, These data suggest that inflammatory cells in the mouse can use a nonoxidative mechanism for microbial killing and permit use of the mouse to study the role such peptides play in host defense and wound repair.