Hirohata Satoshi, Wang Lauren W, Miyagi Masaru, Yan Lin, Seldin Michael F, Keene Douglas R, Crabb John W, Apte Suneel S
Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
J Biol Chem. 2002 Apr 5;277(14):12182-9. doi: 10.1074/jbc.M109665200. Epub 2002 Jan 22.
Punctin (ADAMTSL-1) is a secreted molecule resembling members of the ADAMTS family of proteases. Punctin lacks the pro-metalloprotease and the disintegrin-like domain typical of this family but contains other ADAMTS domains in precise order including four thrombospondin type I repeats. Punctin is the product of a distinct gene on human chromosome 9p21-22 and mouse chromosome 4 that is expressed in adult skeletal muscle. His-tagged punctin expressed in stably transfected High-Five(TM) insect cells was purified to apparent homogeneity by Ni-chromatography of conditioned medium. The NH(2) terminus is not blocked and has the sequence EEDRD and so forth as determined by Edman degradation, demonstrating signal peptidase processing. Recombinant epitope-tagged punctin has a calculated mass of 59,991 Da but exhibits major molecular species of 61970 +/- 6 Da and 62131 +/- 5 Da as measured by liquid chromatography electrospray mass spectrometry. Punctin is a glycoprotein based on carbohydrate staining and liquid chromatography electrospray mass spectrometry glycopeptide analysis. Glycosylation occurs at a single N-linked site as demonstrated by altered electrophoretic migration of punctin expressed in the presence of tunicamycin A. Punctin contains disulfide bonds based on antibody accessibility and electrophoretic migration under reducing versus nonreducing conditions. Rotary shadowing demonstrates that punctin is hatchet-shaped having a globular region attached to a short stem. In transfected COS-1 cells, punctin is deposited in the cell substratum in a punctate fashion and is excluded from focal contacts. Punctin is the first member of a novel family of ADAMTS-like proteins that may have important functions in the extracellular matrix.
穿刺素（ADAMTSL-1）是一种分泌型分子，类似于ADAMTS蛋白酶家族的成员。穿刺素缺乏该家族典型的前金属蛋白酶和类解整合素结构域，但按精确顺序包含其他ADAMTS结构域，包括四个I型血小板反应蛋白重复序列。穿刺素是人类9号染色体p21 - 22区域和小鼠4号染色体上一个独特基因的产物，在成年骨骼肌中表达。在稳定转染的High-Five(TM)昆虫细胞中表达的His标签穿刺素，通过对条件培养基进行镍色谱纯化至表观均一性。通过埃德曼降解法确定，其氨基末端未被封闭，序列为EEDRD等等，表明信号肽已被加工。重组表位标签穿刺素的计算分子量为59,991 Da，但通过液相色谱电喷雾质谱法测定，主要分子种类为61970 +/- 6 Da和62131 +/- 5 Da。根据碳水化合物染色和液相色谱电喷雾质谱糖肽分析，穿刺素是一种糖蛋白。在衣霉素A存在的情况下表达的穿刺素电泳迁移率改变，证明糖基化发生在单个N-连接位点。根据抗体可及性以及还原与非还原条件下的电泳迁移率，穿刺素含有二硫键。旋转阴影显示穿刺素呈斧形，有一个球状区域连接到一个短柄上。在转染的COS-1细胞中，穿刺素以点状方式沉积在细胞基质中，且不包含在黏着斑中。穿刺素是ADAMTS样蛋白新家族的首个成员，可能在细胞外基质中具有重要功能。
