Gene Networks Database

Strongylocentrotus purpuratus Genes in Development: Epidermal growth factor-related proteins

SpEGF III

Function

The SpEGF III gene encodes a member of the fibropellins, EGF repeat-containing proteins that form the apical lamina of the sea urchin embryo.
The size and localization of the SpEGF III protein, and the results of immunoprecipitation assays which reveal that it is tightly associated with the products of SpEGF I, indicate that it is the third major protein component of the apical lamina.
The timing of SpEGF III protein accumulation is coincident with the developmental period when the apical lamina plays an important role in gastrulation.
Genomic Southern blots demonstrated that the SpEGF III is a single copy gene (Bisgrove et al., 1993).

Protein

The SpEGF III cDNA sequence predicts a 570 amino acid protein composed of four distinct structural domains. The first methionine forms the beginning of an 18 amino acid putative signal peptide, in which 11 of the amino acids are hydrophobic. An acceptable cleavage site for signal peptidase (von Heijne, 1984) is present between Thr 17 and Tyr 18.
The signal peptide is followed by a 38 amino acid domain that contains 6 cysteine residues distributed as X4CX4 CX5CX8CXCX8CX2, where X is any residue. This arrangement of cysteines is typical of EGF-like repeat domains and the amino acid sequence of the domain agrees well with the EGF type 2 consensus sequence (Apella et all., 1988).
The 119 amino acid domain following the first EGF-like repeat shows similarity to a C1s-like motif (Journet and Tosi, 1986; Mackinnon et al., 1987)
The internal region of the putative SpEGF III protein contains seven tandemly repeated EGF-like domains of the same size and type as the N terminal EGF-like repeat. Four of the repeats contain the subsequence DXD/N XCXnCXnCXD*/N*X4 Y/FXCXC, which is thought to siganal post-translational hydroxylation of the third Asp and Asn residue (*) of the consensus (Stenflo et al., 1987; Rees et al., 1988). The beta-hydroxylate residue and/or the consensus sequence is thought to play a role in forming a high-affinity calcium-binding site (Rees et al., 1988; Handford et al., 1990). Consensus N-linked glycosylation sites are present within the first and sixth EGF-like repeats.
Carboxy terminal domain of 129 amino acids shares 30% identity with the 128 amino acid adivin protein from chicken egg white (De Lange and Huang, 1971).
The overall structure of the putative SpEGF III protein exactly parallels the predicted structures of the SpEGF I peptides except that the SpEGF III protein tandem EGF-like repeats instead of the 12 or 20 of the alternate splice products of SpEGF I (Delgadillo-Reynoso et al., 1989).
Polyclonal antisera from SpEGF III fusion proteins reveal that the protein is a 100-kDa fibropellin (Bisgrove et al., 1993).
SWISS_PROT: P49013

Subcellular location

Indirect immunofluorescence experiments demonstrated that in 25-hr mesenchyme blastula SpEGF III fibropellin is colocalized with fibropellins Ia and Ib on the surface of cells of the blastoderm (Bisgrove et al., 1993).

Expression Pattern

The 2.9-kb SpEGF III mRNA is is present in unfertilized eggs and throughout embryogenesis and larval development. The relative abundance of SpEGF III mRNA is low (5-10% of maximal) in the unfertilized egg and remains low through the early cleavage stages during the first 12 hr of development. Transcripts begin to show noticeable increase by 16 hr at the late morula stage and then increase dramatically over the next 11 hr reaching maximal levels af accumulation in mesenchyme blastula stage and subseguent stages.
SpEGF III appears to be an embryo-specific transcript as it is present only in adult ovary.
The SpEGF III protein is present in unfertilized eggs but does not reach detectable levels until the mesenchyme blastula stage (27 hr) and then increases dramatically in abundance during gastrulation to reach a maximum level by the prism stage (68 hr). The relative abundance of this protein increases by about 10-fold between 27 and 68 hr.
Localization of fibropellin III is first detectable 2-3 hr after hatching when the vegetal plate has thickened and the primary mesenchyme cells are beginning to ingress. At that time and through subsequent development the protein is localized to the inner surface of the hyaline layer. The localization of fibropellin III in these and later stages is coincident with the localization of SpEGF I fibropellins.
In whole mounts of mesenchyme blastulae and later stages, purified SpEGF III antisera stain a fibrous meshwork that surrounds the embryo. The structure of this matrix changes from a dence, almost amorphous arrangement of small fibers (0.3-0.4 mm) at the mesenchyme blastula stage to a much loosely woven meshwork of larger fibers (0.5-1 mm) in pluteus larvae.
The size and localization of the SpEGF III protein, and the results of immunoprecipitation assays which reveal that it is tightly associated with the products of SpEGF I, indicate that it is the third major protein component of the apical lamina (Bisgrove et al., 1993).

mRNA level

Temporal accumulation

Method: Nothern blot analysis
Reference: Bisgrove et al., 1993

Stage
Unfertilized egg
3 hr
7 hr
12 hr
16 hr
22 hr
25
35 hr
41 hr
51 hr
74 hr
Level *
9
8.5
8
10
24
45
83
82
89
100
75

* Relative levels of the SpEGF III mRNA as determined by densitometry. Values are expressed as the persentage of the maximal level of accumulation.

Protein level

Temporal accumulation

Method: Immunoblot analysis
Reference: Bisgrove et al., 1993

Stage
Unfertilized egg
8 hr (16 cells)
27 hr (mesenchyme blastula)
44 hr (gastrulae)
68 hr (prism)
96 hr (pluteus larvae)
Level
+ -
+ -
+
+
+ +
+ +

Protein spatial localization

Method: Indirect immunofluorescence of 6 mm parafin sections and whole mounts (*) of fixed embryos
Reference: Bisgrove et al., 1993

Stage
2 cells
Hatching blastula
25-hr mesenchyme blastula
42 hr gastrula
68 hr prism *
Level
-
-
surface of cells of the blastoderm
hyaline layer surrounding the embryo and its part which is carried into the archenteron during invagination
staines a fibrous meshwork that surrounds the embryo

Sequences

GenBank:

Regulatory Regions

Regulatory Connections

Upstream Genes

SpEGF III

Downstream Genes

Evolutionary Homologues

Links

Bibliography
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