Funderburgh J L, Funderburgh M L, Hevelone N D, Stech M E, Justice M J, Liu C Y, Kao W W, Conrad G W
Division of Biology, Kansas State University, Manhattan 66506-4901, USA.
Invest Ophthalmol Vis Sci. 1995 Oct;36(11):2296-303.
Lumican is a major proteoglycan of vertebrate cornea. This study characterizes mouse lumican, its molecular form, cDNA sequence, and chromosomal localization.
Lumican sequence was determined from cDNA clones selected from a mouse corneal cDNA expression library using a bovine lumican cDNA probe. Tissue expression and size of lumican mRNA were determined using Northern hybridization. Glycosidase digestion followed by Western blot analysis provided characterization of molecular properties of purified mouse corneal lumican. Chromosomal mapping of the lumican gene (Lcn) used Southern hybridization of a panel of genomic DNAs from an interspecific murine backcross.
Mouse lumican is a 338-amino acid protein with high-sequence identity to bovine and chicken lumican proteins. The N-terminus of the lumican protein contains consensus sequences for tyrosine sulfation. A 1.9-kb lumican mRNA is present in cornea and several other tissues. Antibody against bovine lumican reacted with recombinant mouse lumican expressed in Escherichia coli and also detected high molecular weight proteoglycans in extracts of mouse cornea. Keratanase digestion of corneal proteoglycans released lumican protein, demonstrating the presence of sulfated keratan sulfate chains on mouse corneal lumican in vivo. The lumican gene (Lcn) was mapped to the distal region of mouse chromosome 10. The Lcn map site is in the region of a previously identified developmental mutant, eye blebs, affecting corneal morphology.
This study demonstrates sulfated keratan sulfate proteoglycan in mouse cornea and describes the tools (antibodies and cDNA) necessary to investigate the functional role of this important corneal molecule using naturally occurring and induced mutants of the murine lumican gene.
核纤层蛋白是脊椎动物角膜的主要蛋白聚糖。本研究对小鼠核纤层蛋白及其分子形式、cDNA序列和染色体定位进行了表征。
使用牛核纤层蛋白cDNA探针从小鼠角膜cDNA表达文库中筛选出的cDNA克隆来确定核纤层蛋白序列。使用Northern杂交来确定核纤层蛋白mRNA的组织表达和大小。糖苷酶消化后进行蛋白质印迹分析，以表征纯化的小鼠角膜核纤层蛋白的分子特性。核纤层蛋白基因（Lcn）的染色体定位使用来自种间小鼠回交的一组基因组DNA进行Southern杂交。
小鼠核纤层蛋白是一种338个氨基酸的蛋白质，与牛和鸡的核纤层蛋白具有高度的序列同一性。核纤层蛋白的N端含有酪氨酸硫酸化的共有序列。在角膜和其他几种组织中存在1.9 kb的核纤层蛋白mRNA。抗牛核纤层蛋白抗体与在大肠杆菌中表达的重组小鼠核纤层蛋白发生反应，并且还在小鼠角膜提取物中检测到高分子量蛋白聚糖。角膜蛋白聚糖的角蛋白酶消化释放出核纤层蛋白，表明在体内小鼠角膜核纤层蛋白上存在硫酸化硫酸角质素链。核纤层蛋白基因（Lcn）被定位到小鼠10号染色体的远端区域。Lcn图谱位点位于先前鉴定的影响角膜形态的发育突变体“眼泡”区域。
本研究证明了小鼠角膜中存在硫酸化硫酸角质素蛋白聚糖，并描述了使用小鼠核纤层蛋白基因自然发生和诱导的突变体来研究这种重要角膜分子功能作用所需的工具（抗体和cDNA）。
