Abstract
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.
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).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Colognato, H., MacCarrick, M., O’Rear, J. J., and Yurchenco, P. D. (1997) The laminin alpha2-chain short arm mediates cell adhesion through both the alpha1beta1 and alpha2beta1 Integrins. J. Biol. Chem. 272, 29,330–29,336.
Colognato-Pyke, H., O’Rear, J. J., Yamada, Y., Carbonetto, S., Cheng, Y. S., and Yurchenco. P. D. (1995) Mapping of network-forming, heparin-binding, and alpha 1 beta 1 integrin-recognition sites within the alpha-chain short arm of laminin-1. J. Biol. Chem. 270, 9398–9406.
Fox, J. W., Mayer, U., Nischt, R., Aumailley, M., Reinhardt, D., Wiedemann, H. et al. (1991) Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV. EMBO. J. 10, 3137–3146.
Rambukkana, A., Salzer, J. L., Yurchenco, P. D., and Tuomanen, E. I. (1997) Neural targeting of Mycobacterium leprae mediated by the G domain of the laminin-alpha2 chain. Cell 88, 811–821.
Sung, U., O’Rear, J. J., and Yurchenco, P. D. (1993) Cell and heparin binding in the distal long arm of laminin: identification of active and cryptic sites with recombinant and hybrid glycoprotein [published erratum appears in J. Cell Biol. (1993) Dec;123(6 Pt 1):1623]. J. Cell. Biol. 123, 1255–1268.
Sung, U., O’Rear, J. J., and Yurchenco, P. D., (1997) Localization of heparin binding activity in recombinant laminin G domain. Eur. J. Biochem. 250, 138–43.
Yurchenco, P. D., Quan, Y., Colognato, H., Mathus, T., Harrison, D., Yamada, Y., and O’Rear, J. J. (1997) The alpha chain of laminin-1 is independently secreted and drives secretion of its beta-and gamma-chain partners. Proc. Natl. Acad. Sci. USA 94, 10189–10194.
Yurchenco, P. D., Sung, U., Ward, M. D., Yamada, Y., and O’Rear, J. J. (1993) Recombinant laminin G domain mediates myoblast adhesion and heparin binding. J. Biol. Chem. 268, 8356–8365.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Humana Press Inc.
About this protocol
Cite this protocol
Mathus, T.L., Yurchenco, P.D. (2000). Analysis of Laminin Structure and Function with Recombinant Glycoprotein Expressed in Insect Cells. In: Streuli, C.H., Grant, M.E. (eds) Extracellular Matrix Protocols. Methods in Molecular Biology™, vol 139. Humana Press. https://doi.org/10.1385/1-59259-063-2:27
Download citation
DOI: https://doi.org/10.1385/1-59259-063-2:27
Publisher Name: Humana Press
Print ISBN: 978-0-89603-624-6
Online ISBN: 978-1-59259-063-6
eBook Packages: Springer Protocols