Recombinant laminin G domain mediates myoblast adhesion and heparin binding.
A recombinant mouse cDNA fragment encoding the G domain of the basement membrane laminin A chain was inserted into the eukaryotic baculovirus expression vector pVL1392 modified to produce fusion proteins carrying the rat fibronectin signal. G domain, expressed and secreted as a soluble glycoprotein (rG), was purified to near homogeneity without denaturing conditions. By electron microscopy rG possessed the same globular morphology as found in laminin. rG was cleaved with elastase into two fragments, rG70 and rG50. The latter fragment possessed the identical N terminus as laminin fragment E3 and both shared the same secondary structure by circular dichroism. rG, and rG containing a deletion (residues 2980-3028) rich in basic residues bound to heparin with similar avidity. rG also promoted mouse C2 myoblast cell adhesion and spreading, and evaluation of myoblasts on rG70 and rG50 further revealed that cell spreading was an activity confined to the more proximal sequence of rG70. Antibody specific for rG70 completely blocked cell adhesion to intact laminin in contrast to antibody specific for E3. Finally rG did not inhibit laminin polymerization. These data support the role of G domain in cell and heparin binding, but not laminin self-assembly, and the approach provides a means to further characterize these functions.
Yurchenco
P D
PD
Department of Pathology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
Sung
U
U
Ward
M D
MD
Yamada
Y
Y
O'Rear
J J
JJ
D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D016367:Baculoviridae / Q000235:genetics; D002448:Cell Adhesion / Q000187:drug effects*; D002460:Cell Line; D002942:Circular Dichroism; D005353:Fibronectins / Q000235:genetics; D005822:Genetic Vectors; D006493:Heparin / Q000378:metabolism*; D007797:Laminin / Q000737:chemistry / Q000494:pharmacology* / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron; D009036:Moths; D009132:Muscles / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010196:Pancreatic Elastase; D010446:Peptide Fragments / Q000302:isolation & purification / Q000494:pharmacology*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology / Q000648:ultrastructure; D017384:Sequence Deletion; D014162:Transfection
D000906:Antibodies; D005353:Fibronectins; D007797:Laminin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006493:Heparin; D010196:Pancreatic Elastase