Regulation of biological activity and matrix assembly of laminin-5 by COOH-terminal, LG4-5 domain of alpha3 chain.
The basement membrane protein laminin-5 (LN5; alpha3beta3gamma2) undergoes specific proteolytic processing of the 190-kDa alpha3 chain to the 160-kDa form after the secretion, releasing its COOH-terminal, LG4-5 domain. To clarify the biological significance of this processing, we tried to express a recombinant precursor LN5 with a 190-kDa alpha3 chain (pre-LN5), in which the cleavage sequence Gln-Asp was changed to Ala-Ala by point mutation. When the wild-type and mutated LN5 heterotrimers were expressed in HEK293 cells, the wild-type alpha3 chain was completely cleaved, whereas the mutated alpha3 chain was partially cleaved at the same cleavage site (Ala-Ala). pre-LN5 was preferentially deposited on the extracellular matrix, but this deposition was effectively blocked by exogenous heparin. This suggests that interaction between the LG4-5 domain and heparan sulfate proteoglycans on the cell surface and/or extracellular matrix is important in the matrix assembly of LN5. Next, we purified both pre-LN5 and the mature LN5 with the processed, 160-kDa alpha3 chain (mat-LN5) from the conditioned medium of the HEK293 cells and compared their biological activities. mat-LN5 showed higher activities to promote cell adhesion, cell scattering, cell migration, and neurite outgrowth than pre-LN5. These results indicate that the proteolytic removal of LG4-5 from the 190-kDa alpha3 chain converts the precursor LN5 from a less active form to a fully active form. Furthermore, the released LG4-5 fragment stimulated the neurite outgrowth in the presence of mat-LN5, suggesting that LG4-5 synergistically enhances integrin signaling as it is released from the precursor LN5.
Tsubota
Yoshiaki
Y
Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Japan.
Yasuda
Chie
C
Kariya
Yoshinobu
Y
Ogawa
Takashi
T
Hirosaki
Tomomi
T
Mizushima
Hiroto
H
Miyazaki
Kaoru
K
D000409:Alanine / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000096:biosynthesis* / Q000502:physiology; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000378:metabolism; D015723:Gene Library; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry; D006493:Heparin / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D015603:Keratinocytes / Q000378:metabolism; D007797:Laminin / Q000737:chemistry; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D016716:PC12 Cells; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014945:Wound Healing; D000097794:Kalinin
D000409:Alanine; D015815:Cell Adhesion Molecules; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin; D007797:Laminin; D011994:Recombinant Proteins; D000097794:Kalinin; C095567:laminin alpha 3