RESUMO
Neural epidermal growth factor-like (NEL)-like 1 (NELL1) is a secretory osteogenic protein comprising an N-terminal thrombospondin-1-like (TSPN) domain, four von Willebrand factor type C domains, and six epidermal growth factor-like repeats. NELL1 shows heparin-binding activity; however, the biological significance remains to be explored. In this report, we demonstrate that NELL1 binds to cell surface proteoglycans through its TSPN domain. Major heparin-binding sites were identified on the three-dimensional structural model of the TSPN domain of NELL1. Mutant analysis of the heparin-binding sites indicated that the heparin-binding activity of the TSPN domain is involved in interaction of NELL1 with cell surface proteoglycans.
Assuntos
Anticoagulantes/metabolismo , Membrana Celular/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Heparina/metabolismo , Modelos Moleculares , Proteínas do Tecido Nervoso/metabolismo , Osteoblastos/metabolismo , Animais , Sítios de Ligação , Proteínas de Ligação ao Cálcio , Linhagem Celular , Humanos , Cinética , Células-Tronco Mesenquimais/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da Espécie , Trombospondina 1/metabolismoRESUMO
NELL1 is a large oligomeric secretory glycoprotein that functions as an osteoinductive factor. NELL1 contains several conserved domains, has structural similarities to thrombospondin 1, and supports osteoblastic cell adhesion through integrins. To define the structural requirements for NELL1-mediated cell adhesion, we prepared a series of recombinant NELL1 proteins (intact, deleted, and cysteine-mutant) from a mammalian expression system and tested their activities. A deletion analysis demonstrated that the C-terminal cysteine-rich region of NELL1 is critical for the cell adhesion activity of NELL1. Reducing agent treatment decreased the cell adhesion activity of full-length NELL1 but not of its C-terminal fragments, suggesting that the intramolecular disulfide bonds within this region are not functionally necessary but that other disulfide linkages in the N-terminal region of NELL1 may be involved in cell adhesion activity. By replacing cysteine residues with serines around the coiled-coil domain of NELL1, which is responsible for oligomerization, we created a mutant NELL1 protein that was unable to form homo-oligomers, and this monomeric mutant showed substantially lower cell adhesion activity than intact NELL1. These results suggest that an oligomerization-induced conformational change in the C-terminal region of NELL1 is important for the efficient mediation of cell adhesion and spreading by NELL1.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Movimento Celular/fisiologia , Glicoproteínas/metabolismo , Multimerização Proteica/fisiologia , Animais , Proteínas de Ligação ao Cálcio/genética , Adesão Celular/fisiologia , Linhagem Celular , Glicoproteínas/genética , Camundongos , Mutação , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
NELL1 is a secretory osteogenic protein containing several structural motifs that suggest that it functions as an extracellular matrix component. To determine the mechanisms underlying NELL1-induced osteoblast differentiation, we examined the cell-adhesive activity of NELL1 using a series of recombinant NELL1 proteins. We demonstrated that NELL1 promoted osteoblastic cell adhesion through at least three cell-binding domains located in the C-terminal region of NELL1. Adhesion of cells to NELL1 was strongly inhibited by function-blocking antibodies against integrin α3 and ß1 subunits, suggesting that osteoblastic cells adhered to NELL1 through integrin α3ß1. Further, focal adhesion kinase activation is involved in NELL1 signaling.
