Identification of neurite outgrowth promoting sites on the laminin alpha 3 chain G domain.
Laminins are expressed in specific tissues and are involved in various biological activities including promoting cell adhesion, growth, migration, neurite outgrowth, and differentiation. The laminin alpha3 chain is mainly located in the skin and is also expressed in the floor plate of the developing neural tube. Previously, we showed that the human laminin alpha3 chain LG4 module binds to syndecan-2/4, a membrane-associated proteoglycan, and promotes human fibroblast adhesion. Here, we have evaluated the neurite outgrowth activity of the laminin alpha3 chain LG4 and LG5 modules. Three overlapping recombinant proteins, which contained LG4 and/or LG5 modules of the human laminin alpha3 chain, were prepared using a mammalian cell expression system. Two proteins, rec-alpha3LG4-5 and rec-alpha3LG4, promoted cell attachment and neurite outgrowth of rat pheochromocytoma PC12 cells, but rec-alpha3LG5 was inactive. Twenty-two peptides covering the entire LG4 module were synthesized and tested for cell attachment and neurite outgrowth activity to identify active sites of the LG4 module. A3G75 (KNSFMALYLSKG, alpha3 chain 1411-1422) and A3G83 (GNSTISIRAPVY, alpha3 chain 1476-1487) promoted PC12 cell attachment and neurite outgrowth. Additionally, A3G75 and A3G83 inhibited PC12 cell attachment to rec-alpha3LG4. These results suggest that the A3G75 and A3G83 sites are important for PC12 cell attachment and neurite outgrowth in the laminin alpha3 chain LG4 module. We also conjugated the A3G75 and A3G83 peptides on chitosan membranes to test their potential as bio-materials. These peptide-conjugated chitosan membranes were more active for neurite outgrowth than the peptide-coated plates. These results suggest that the A3G75- and A3G83-conjugated chitosan membranes are applicable as bio-medical materials for neural tissue repair and engineering.
Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan.
Kozue
K
Kato
Atsushi
A
Utani
Nobuharu
N
Suzuki
Mayumi
M
Mochizuki
Masanori
M
Yamada
Norio
N
Nishi
Hiroshi
H
Matsuura
Hiroshi
H
Shinkai
Motoyoshi
M
Nomizu
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001704:Biopolymers / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002686:Chitin / Q000031:analogs & derivatives / Q000494:pharmacology; D048271:Chitosan; D006801:Humans; D007797:Laminin / Q000737:chemistry / Q000502:physiology*; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000737:chemistry / Q000502:physiology*; D016501:Neurites / Q000187:drug effects / Q000502:physiology*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000494:pharmacology; D016716:PC12 Cells; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology
D001704:Biopolymers; D007797:Laminin; D008567:Membranes, Artificial; D009414:Nerve Growth Factors; D009842:Oligopeptides; D011994:Recombinant Proteins; D002686:Chitin; C095567:laminin alpha 3; D048271:Chitosan