Cell type-specific differences in glycosaminoglycans modulate the biological activity of a heparin-binding peptide (RKRLQVQLSIRT) from the G domain of the laminin alpha1 chain.
AG73 (RKRLQVQLSIRT), a peptide from the G domain of the laminin alpha1 chain, has diverse biological activities with different cell types. The heparan sulfate side chains of syndecan-1 on human salivary gland cells were previously identified as the cell surface ligand for AG73. We used homologous peptides from the other laminin alpha-chains (A2G73-A5G73) to determine whether the bioactivity of the AG73 sequence is conserved. Human salivary gland cells and a mouse melanoma cell line (B16F10) both bind to the peptides, but cell attachment was inhibited by glycosaminoglycans, modified heparin, and sized heparin fragments in a cell type-specific manner. In other assays, AG73, but not the homologous peptides, inhibited branching morphogenesis of salivary glands and B16F10 network formation on Matrigel. We identified residues critical for AG73 bioactivity using peptides with amino acid substitutions and truncations. Fewer residues were critical for inhibiting branching morphogenesis (XKXLXVXXXIRT) than those required to inhibit B16F10 network formation on Matrigel (N-terminal XXRLQVQLSIRT). In addition, surface plasmon resonance analysis identified the C-terminal IRT of the sequence to be important for heparin binding. Structure-based sequence alignment predicts AG73 in a beta-sheet with the N-terminal K (Lys(2)) and the C-terminal R (Arg(10)) on the surface of the G domain. In conclusion, we have determined that differences in cell surface glycosaminoglycans and differences in the amino acids in AG73 recognized by cells modulate the biological activity of the peptide and provide a mechanism to explain its cell-specific activities.
Hoffman
M P
MP
Craniofacial Developmental Biology and Regeneration Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4370, USA. mhoffman@mail.nih.gov
Engbring
J A
JA
Nielsen
P K
PK
Vargas
J
J
Steinberg
Z
Z
Karmand
A J
AJ
Nomizu
M
M
Yamada
Y
Y
Kleinman
H K
HK
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001710:Biotin / Q000378:metabolism; D002448:Cell Adhesion; D018360:Crystallography, X-Ray; D006025:Glycosaminoglycans / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D007797:Laminin / Q000378:metabolism*; D051379:Mice; D008813:Mice, Inbred ICR; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation; D020349:Surface Plasmon Resonance
C105786:AG 73; D006025:Glycosaminoglycans; D007797:Laminin; D010446:Peptide Fragments; D001710:Biotin; D006493:Heparin