TitleIdentification and characterization of critical laminin-111 sequences required for polymerization and cell surface anchorage and the requirement of both activities for proper basement membrane formation and other events
NameHarrison, David A. (author), Amenta, Peter S (chair), Yurchenco, Peter D (internal member), Carr, Chavela M (internal member), Sahota, Amrik (outside member), Rutgers University, Graduate School - New Brunswick,
DescriptionA system was developed for expression and purification of various recombinant a1 LG4-5 and heterotrimeric laminin-111 proteins. A number of combinations involving different promoters, 5’UTRs, signal sequences, epitope tags, selectable antibiotics, and purification schemes were tested in order to optimize the system. A homology model of laminin a1 LG4-5 was generated and utilized to identify candidates for mutation and expression as recombinant a1 LG4-5 proteins. The crystal structure of a1 LG4-5 was also determined. Heparin, a-dystroglycan (aDG), and sulfatide binding of the generated mutants demonstrated wide differences and dependence upon contributions from basic residues on the surface of LG4, including: RKR2721 and KRK2793 for heparin; RAR2833, KDR2860, the Asn2811 glycation moiety, and the LG4 Ca2+ ion for aDG; the LG4 Ca2+, Arg2833 of RAR2833, and Lys2766 residue of RKGRTK2770 for one sulfatide binding site, and the Arg2831 of RAR2833 for a second sulfatide site. The produced recombinant heterotrimeric laminin-111s demonstrated a requirement for the N-terminal LN domains of laminin-111’s constituent a1, b1, and g1 chains in self-polymerization. The inability of embryoid bodies, derived from laminin g1 null embryonic stem cells, to express Lm-111 and develop past formation of an outer endodermal layer of cells without laminin, was utilized via the addition of exogeneous recombinant laminin-111s, to test the various functions of the recombinant laminins and their ability to form a basement membrane and induce both differentiation of an epiblast layer and formation of a central cavity. Laminins’ containing defective polymerization or a1 LG4 anchorage failed to form a BM or undergo any further development after endodermal differentiation. Experiments with Schwann cells, C2C12 myotubes, and mouse embryonic fibroblasts demonstrated the requirement for both polymerization and a1 LG4 mediated anchorage in laminin-111 for proper BM formation, cytoskeletal attachment, and laminin induced cell signaling via Src. The results also suggest a role for laminin-111 polymerization and anchorage in providing a means for aggregation and accumulation of low affinity interactions in a complex, thereby attaining very high net affinities for binding interactions and attaining otherwise unatainable thresholds necessary for these interactions to exert their effects through manipulation of the cytoskeleton, as well as, laminin induced differentiation and cell signaling.
NoteIncludes bibliographical references
Noteby David A. Harrison
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.