Analysis of Laminin Structure and Function with Recombinant Glycoprotein Expressed in Insect Cells

Recent developments in the application of eukaryotic recombinant protein techniques have provided new tools with which to dissect and map functional activities in basement membrane glycoproteins. This has been particularly valuable in the case of laminins where the relationship between structure and function has been difficult to establish. Several characteristics of the laminins lie at the heart of the problem. First, the molecules are very large, each assembled from three multidomain subunits joined in a long-coiled coil Fig. 1 ). Second, laminins bear substantial disulfide and carbohydrate modifications that are crucial for proper conformation. Many, perhaps most, laminin activities present in native laminin are lost upon heat- or chaotropic-denaturation. Native activities are often not retained in short peptides or even in recombinant fragments generated in prokaryotic cells. Furthermore, there is evidence to suggest that synthetic laminin peptides can exhibit “cryptic” activities not found in native protein.   Fig. 1.  Structure and functions of laminin-1 and it’s proteolytic derivatives. Laminins are heterotrimeric glycoproteins composed of α, β, and γ chains joined in a coiled-coil to form a cruciform-shaped molecule. The carboxyl-terminal G (globular) domain of the α chain extends from the coiled-coil. Roman numerals indicate domains that are found within each subunit chain. Laminin-1 can support integrin-mediated adhesion of cells (α1β1, α2β1, α6β1, α6β4, α7β1), undergo self-polymerization (polymer), and bind entactin/nidogen (En/Nd), α-dystroglycan (αDG), and heparin. Note that several domains have overlapping activities. Recombinant G domain (rG) and recombinant G domain with proximal α chain coiled-coil (rAiG) have been purified and shown to have activities similar to those found in native laminin-1 purified from tissue ( 5 , 6 , 8 ).