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The structure of the Ca²+-binding, glycosylated F-spondin domain of F-spondin - A C2-domain variant in an extracellular matrix protein.

Tan K, Lawler J - BMC Struct. Biol. (2011)

Bottom Line: The FS domain is found in F-spondins, mindins, M-spondin and amphiF-spondin.The unique feature of F-spondin FS domain is the presence of three disulfide bonds associated with the N- and C-termini of the domain and a highly conserved N-linked glycosylation site.The integrin-binding motif found in mindin is not conserved in the F-spondin FS domain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA. ktan@anl.gov

ABSTRACT

Background: F-spondin is a multi-domain extracellular matrix (ECM) protein and a contact-repellent molecule that directs axon outgrowth and cell migration during development. The reelin_N domain and the F-spondin domain (FS domain) comprise a proteolytic fragment that interacts with the cell membrane and guides the projection of commissural axons to floor plate. The FS domain is found in F-spondins, mindins, M-spondin and amphiF-spondin.

Results: We present the crystal structure of human F-spondin FS domain at 1.95Å resolution. The structure reveals a Ca2+-binding C2 domain variant with an 8-stranded antiparallel β-sandwich fold. Though the primary sequences of the FS domains of F-spondin and mindin are less than 36% identical, their overall structures are very similar. The unique feature of F-spondin FS domain is the presence of three disulfide bonds associated with the N- and C-termini of the domain and a highly conserved N-linked glycosylation site. The integrin-binding motif found in mindin is not conserved in the F-spondin FS domain.

Conclusion: The structure of the F-spondin FS domain completes the structural studies of the multiple-domain ECM molecule. The homology of its core structure to a common Ca2+- and lipid-binding C2 domain suggests that the F-spondin FS domain may be responsible for part of the membrane targeting of F-spondin in its regulation of axon development. The structural properties of the FS domain revealed in this study pave the way for further exploration into the functions of F-spondin.

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The multiple domain structure of F-spondin and FS domain expression. (A) Schematic diagram of the domain structure of F-spondin showing the disulfide bond patterns of each domain and highly charged linkers between FS domain and TSR1, and between TSR5 and TSR6. (B) Size-exclusion chromatography of the recombinant FS domain. The recombinant protein was first purified using Ni-affinity chromatography. In the following FPLC runs with a Superdex™ 75 column (GE Healthcare Life Sciences), six protein standards were used to calibrate the column (data not shown). In the run of FS domain, the molecular mass of the principal peak was calculated to be 22.7 kDa, which was smaller than the calculated molecular mass of 27.23 kDa that does not include vector-derived sequences and N-linked glycans. SDS PAGE of the FS domain revealed a single band with a molecular mass greater than 30 kDa (insert).
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Figure 1: The multiple domain structure of F-spondin and FS domain expression. (A) Schematic diagram of the domain structure of F-spondin showing the disulfide bond patterns of each domain and highly charged linkers between FS domain and TSR1, and between TSR5 and TSR6. (B) Size-exclusion chromatography of the recombinant FS domain. The recombinant protein was first purified using Ni-affinity chromatography. In the following FPLC runs with a Superdex™ 75 column (GE Healthcare Life Sciences), six protein standards were used to calibrate the column (data not shown). In the run of FS domain, the molecular mass of the principal peak was calculated to be 22.7 kDa, which was smaller than the calculated molecular mass of 27.23 kDa that does not include vector-derived sequences and N-linked glycans. SDS PAGE of the FS domain revealed a single band with a molecular mass greater than 30 kDa (insert).

Mentions: F-spondin was initially identified in the rat embryo floor plate[1], a ventralizing structure implicated in the control of neural cell patterning and axon growth in the developing vertebrate nervous system. The expression level of F-spondin is high in the floor plate at the time of axon extension[1]. Further studies have found that F-spondin plays an important role in the outgrowth of sensory[1], commissural[2] and hippocampal[3] neurons during development as well as in the migration of distinct somite domains to neural crest[4]. F-spondin is an extracellular matrix (ECM) protein with multiple domains, including an N-terminal domain (reelin_N domain)[5], an F-spondin (FS) domain and six thrombospondin type 1 repeats (TSRs) (Figure 1A). F-spondin, which is secreted by cells within the floor plate, is proteolytically processed into fragments that differentially bind to the floor plate cells or the basement membrane[6]. The portion that binds to the floor plate cells acts as a short-range repellent of commissural axons and prevents their migration into the floor plate. Other portions of F-spondin that includes the FS domain accumulate at the basement membrane and support growth cone migration[6]. The proteolysis of F-spondin and the coordinated interaction of the different fragments with the membrane of floor plate and the basement membrane provide a combinatorial guidance signal for commissural axons that cross the midline[6]. F-spondin is also highly up-regulated in injured peripheral nerves and it promotes outgrowth of sensory neurons[2]. An antibody against the FS domain blocks neurite outgrowth, indicating the FS domain plays an active role in axon regeneration[2]. This observation is largely in agreement with the fact that an F-spondin fragment that lacks the TSRs is sufficient to promote neuronal differentiation[7].


The structure of the Ca²+-binding, glycosylated F-spondin domain of F-spondin - A C2-domain variant in an extracellular matrix protein.

Tan K, Lawler J - BMC Struct. Biol. (2011)

The multiple domain structure of F-spondin and FS domain expression. (A) Schematic diagram of the domain structure of F-spondin showing the disulfide bond patterns of each domain and highly charged linkers between FS domain and TSR1, and between TSR5 and TSR6. (B) Size-exclusion chromatography of the recombinant FS domain. The recombinant protein was first purified using Ni-affinity chromatography. In the following FPLC runs with a Superdex™ 75 column (GE Healthcare Life Sciences), six protein standards were used to calibrate the column (data not shown). In the run of FS domain, the molecular mass of the principal peak was calculated to be 22.7 kDa, which was smaller than the calculated molecular mass of 27.23 kDa that does not include vector-derived sequences and N-linked glycans. SDS PAGE of the FS domain revealed a single band with a molecular mass greater than 30 kDa (insert).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 1: The multiple domain structure of F-spondin and FS domain expression. (A) Schematic diagram of the domain structure of F-spondin showing the disulfide bond patterns of each domain and highly charged linkers between FS domain and TSR1, and between TSR5 and TSR6. (B) Size-exclusion chromatography of the recombinant FS domain. The recombinant protein was first purified using Ni-affinity chromatography. In the following FPLC runs with a Superdex™ 75 column (GE Healthcare Life Sciences), six protein standards were used to calibrate the column (data not shown). In the run of FS domain, the molecular mass of the principal peak was calculated to be 22.7 kDa, which was smaller than the calculated molecular mass of 27.23 kDa that does not include vector-derived sequences and N-linked glycans. SDS PAGE of the FS domain revealed a single band with a molecular mass greater than 30 kDa (insert).
Mentions: F-spondin was initially identified in the rat embryo floor plate[1], a ventralizing structure implicated in the control of neural cell patterning and axon growth in the developing vertebrate nervous system. The expression level of F-spondin is high in the floor plate at the time of axon extension[1]. Further studies have found that F-spondin plays an important role in the outgrowth of sensory[1], commissural[2] and hippocampal[3] neurons during development as well as in the migration of distinct somite domains to neural crest[4]. F-spondin is an extracellular matrix (ECM) protein with multiple domains, including an N-terminal domain (reelin_N domain)[5], an F-spondin (FS) domain and six thrombospondin type 1 repeats (TSRs) (Figure 1A). F-spondin, which is secreted by cells within the floor plate, is proteolytically processed into fragments that differentially bind to the floor plate cells or the basement membrane[6]. The portion that binds to the floor plate cells acts as a short-range repellent of commissural axons and prevents their migration into the floor plate. Other portions of F-spondin that includes the FS domain accumulate at the basement membrane and support growth cone migration[6]. The proteolysis of F-spondin and the coordinated interaction of the different fragments with the membrane of floor plate and the basement membrane provide a combinatorial guidance signal for commissural axons that cross the midline[6]. F-spondin is also highly up-regulated in injured peripheral nerves and it promotes outgrowth of sensory neurons[2]. An antibody against the FS domain blocks neurite outgrowth, indicating the FS domain plays an active role in axon regeneration[2]. This observation is largely in agreement with the fact that an F-spondin fragment that lacks the TSRs is sufficient to promote neuronal differentiation[7].

Bottom Line: The FS domain is found in F-spondins, mindins, M-spondin and amphiF-spondin.The unique feature of F-spondin FS domain is the presence of three disulfide bonds associated with the N- and C-termini of the domain and a highly conserved N-linked glycosylation site.The integrin-binding motif found in mindin is not conserved in the F-spondin FS domain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA. ktan@anl.gov

ABSTRACT

Background: F-spondin is a multi-domain extracellular matrix (ECM) protein and a contact-repellent molecule that directs axon outgrowth and cell migration during development. The reelin_N domain and the F-spondin domain (FS domain) comprise a proteolytic fragment that interacts with the cell membrane and guides the projection of commissural axons to floor plate. The FS domain is found in F-spondins, mindins, M-spondin and amphiF-spondin.

Results: We present the crystal structure of human F-spondin FS domain at 1.95Å resolution. The structure reveals a Ca2+-binding C2 domain variant with an 8-stranded antiparallel β-sandwich fold. Though the primary sequences of the FS domains of F-spondin and mindin are less than 36% identical, their overall structures are very similar. The unique feature of F-spondin FS domain is the presence of three disulfide bonds associated with the N- and C-termini of the domain and a highly conserved N-linked glycosylation site. The integrin-binding motif found in mindin is not conserved in the F-spondin FS domain.

Conclusion: The structure of the F-spondin FS domain completes the structural studies of the multiple-domain ECM molecule. The homology of its core structure to a common Ca2+- and lipid-binding C2 domain suggests that the F-spondin FS domain may be responsible for part of the membrane targeting of F-spondin in its regulation of axon development. The structural properties of the FS domain revealed in this study pave the way for further exploration into the functions of F-spondin.

Show MeSH