Sung U, O'Rear J J, Yurchenco P D
Department of Pathology, Robert Wood Johnson Medical School and University of Medicine and Dentistry of New Jersey, Piscataway, USA.
Eur J Biochem. 1997 Nov 15;250(1):138-43. doi: 10.1111/j.1432-1033.1997.00138.x.
Basement membrane laminin (laminin-1) is a multidomain glycoprotein that interacts with itself, heparin and cells. The interaction with heparin/heparan sulfate proteglycans is thought to be important for the architectural formation of basement membranes and adhesion to cells. The major heparin binding site has been known to reside in the long arm globular domain (G domain). The G domain is in turn subdivided into five subdomains (G1-G5). In order to localize the heparin binding regions further, recombinant G domains (rG and rG5) were expressed in Sf9 insect cells using baculovirus expression vector. By the limited proteolysis of recombinant G domains followed by either heparin affinity HPLC or overlay with radiolabeled heparin, the relative affinity of each subdomain to heparin was assigned as G1>G2 = G4>G5>G3, such that G1 bound strongly and G3 not at all. Since the activity in G1-G3 is cryptic in intact laminin long arm [Sung, U., O'Rear, J. J. & Yurchenco, P. D. (1993) J. Cell Biol. 123, 1255-1268], the active heparin binding site of G domain appears to be located in G4 and proximal G5.
基底膜层粘连蛋白（层粘连蛋白-1）是一种多结构域糖蛋白，可与自身、肝素及细胞相互作用。与肝素/硫酸乙酰肝素蛋白聚糖的相互作用被认为对基底膜的结构形成及与细胞的黏附很重要。已知主要的肝素结合位点位于长臂球状结构域（G结构域）。G结构域又进一步细分为五个亚结构域（G1-G5）。为了进一步定位肝素结合区域，使用杆状病毒表达载体在Sf9昆虫细胞中表达了重组G结构域（rG和rG5）。通过对重组G结构域进行有限蛋白酶解，随后进行肝素亲和高效液相色谱或与放射性标记肝素进行覆盖分析，每个亚结构域与肝素的相对亲和力被确定为G1>G2 = G4>G5>G3，即G1结合力强，G3完全不结合。由于在完整的层粘连蛋白长臂中G1-G3的活性是隐蔽的[宋，U.，奥里尔，J. J. & 尤尔琴科，P. D.（1993年）《细胞生物学杂志》123，1255 - 1268]，G结构域的活性肝素结合位点似乎位于G4和近端G5。
