BP10

Function

BP10 codes for a metalloprotease which belongs to huBMP-1 related protein family.
It plays a role in the differentiation and morphogenesis of ectodermal lineages and subsequent patterning of the embryo. The BP10 protein and its mRNA are molecular markers of the presumptive ectoderm territory (Lepage et al., 1992).

Protein

BP10 protein contains seven domains: a signal peptide, a putative action peptide, a catalytic domain with an active center typical of a Zn^2+ metalloprotease, an EGF-like domain and two internal repeats similar to repeated domains found in the C1s and C1r serine proteases of the complement cascade.
Protein contains 597 amino acids. it is a hydrophilic protein with an excess of acidic (81 Asp + Glu) over basic amino acids (63 Arg + Lys + His) and unusually high number of methionine and cysteine residues. N-terminal sequence has two plausible cleavage sites with high scores, between residues Cys 16 and Thr 17 and between Ala 19 and Ala 20. The first site is clearly favored over the second one and thus Thr 17 will be taken provisionally as the first residue of the secreted protein.
Most of the cysteine residues are found in the C-terminal half of the molecule and 8 of them are very close to each other in a small cysteine-rich region between residues 293 and 339. No potential N-glycosylation site was found but there are series of 25 amino acids containing 14 threonines grouped in five clusters of 2-3 residues at positions 451-475.
The threonine-rich region splits the terminal half of the protein into 2 regions which have almost exactly the same length, each containing 4 cysteine residues at the same positions and sharing 35 % identity. These threonine clusters may be the site of O-glycosylations.
The C-terminal end of the BP10 protein is thus made up of two homologous repeats of the same domain, arranged in tandem. And there's also a basic stretch RKKRKA at positions 88-93, whose sequence is reminiscent of those present in some precursors recognized by intracellular processing enzymes with a trypsin-like specificity (Bentley et al., 1986).
A 200 amino acid domain of the BP10 protein, beginning around position 90 and extending to about residue 295, is homologous to a whole small digestive protease of 22 * 10^3, isolated from the crayfish (Astacus), which is a classical zinc-dependent metalloenzyme (Titani et al., 1987; Stocker et al., 1988). The overall homology score is 40 % in a 200 amino acid overlap. A sequence homologous to this domain was also found in the human protein BMP-1. 42 % of the 200 amino acid residues of the BP10 and BMP-1 domains are identical. The positions of the four cysteines of the Astacus protease are conserved in the BP10 and BMP-1, suggesting that these domains are similarly folded. The N-terminal of the crayfish protease, determined for the purified active enzyme, aligns with position 94 in the BP10 sequence and position 121 in the BMP-1 sequence, just downstream of the basic peptides RKKRK (BP10) and RSRSRR (BMP-1) which resemble many cleavage sites by intracellular processing enzymes. This suggest that, like most of the proteolytic enzymes, these proteases might be synthesized as precursors and later processed by proteolytic cleavage (Lepage et al., 1992).
SWISS_PROT: P42674

Subcellular location

At the early blastula stage, the protein is present in a diffuse pattern in the perinuclear region suggesting that it is synthesized in the endoplasmic reticulum surrounding the nuclei.
BP10 is predicted to be a secreted protein. Immunolocalization of the BP10 protein in whole embryos (and in individual cells from dissociated embryos) clearly showed that in the hatching blastulas, the BP10 protein is transported towards the apical face of the epithelial cells, and can be seen just beneath the membrane in older blastulas. As the predicted protein sequence does not contain any putative hydrophobic transmembrane spanning region, it is likely that BP10 is secreted into the perivitelline space. Thus, the BP10 protein is synthesized and secreted by ectoderm cells and probably acts on the surface of the embryo (Lepage et al., 1992).

Expression Pattern

The BP10 mRNA is a zygotic gene product synthesized in the very first hours following fertilization and activation of the zygotic genome. The 2.9 kb BP10 transcript begins to accumulate at the early blastula stage (about 128 cells), reaches its highest level at the prehatching blastula stage (8 hours, about 200 cells per embryo), then decreases to an undetectable level when the blastula is hatched.
The BP10 protein is not detectable either in the unfertilized egg, which indicates that there is no maternal BP10 protein, or in the very early cleavage stages. It begins to be detected, albeit weakly, at the early blastula stage (about 250 cells), its abundance peaks at the prehatching and swimming blastula stages then decreases rapidly and remains constant throughout development until the prism stage which was the last analysed.
In all the labelled embryos, the BP10 staining is restricted to a domain covering about half the embryo. The localization of the BP10 protein correlates with the distribution of its mRNA. Then it can be detected in an apical and submembranous position just before its secretion into the perivitelline space. The protein is synthesized in a sharply delimited continuous territory spanning about 70 % of the blastula. Comparison of the size and orientation of the labelled territory in the late blastula with the fate map of the blastula stage embryo shows that the domain in which the BP10 gene is expressed corresponds to the presumptive ectoderm (Lepage et al., 1992).

mRNA level

Temporal accumulation
Method 1: Northern blot analysis
Reference: Lepage et al., 1992
Method 2: Run-on assays
Reference: Lepage et al., 1992

Stage Egg 2 hr 4 hr 6.5 hr 7.5 hr 8.5 hr 9.5 hr 10.5 hr 11.5 hr 12.5 hr 15 hr 30 hr 60 hr

Level - - - + + + + + + + - - - -

Protein level

Temporal accumulation
Method: Western blot analysis
Reference: Lepage et al., 1992

Stage Egg 16 cells Early blastula Prehatching blastula Swimming blastula Mesenchyme blastula Gastrula Prism

Level - - - + + + + +

Ectopic expression

Dissociated blastomeres
Expression of BP10 transcripts
Slot-blot hybridization showed that the pattern seen in intact embryos was identical with the pattern described above.
In blastomers raised permanently in isolation, the mRNA level increased with the same time course as intact embryos and reached the ame peak value. However, where in normal embryos the transcripts disappear rapidly, in isolated blastomeres their level begins to decrease but then remains constant for many hours.
The abnormal pattern observed is independent of the association state of blastomeres during the early stages and is not due to a prolonged Ca²⁺ deprivation and cannot be corrected by Ca²⁺ addition.
Thus the increase in transcripts level is indepenent of contacts and short-range interactions between blastomeres.

Synthesis of BP10 protein
Blastomers were raised in isolation for ~ 10 h and stained with antibodies directed against BP10. The immunolabeling of individual blastomers resembled that observed in whole blastulae: a single spot was visible on one side of the nucleus.
Furthermore, only two-thirds of the blastomeres were labeled. This corresponds to the fraction of cells which synthesises the protein in intact embryos, suggesting thta the same cells were labelled in both cases. Thus, dissociation does not affect the inherent spatial control of this gene (Ghiglione et al., 1993)

Action of animalizing agents
Expression of BP10 transcripts
Slot-blot hybrydization showed that in embryos treated with Zn²⁺, the time course of accumulation and decay as well as the maximal abundance were almost identical to those in the control embryos.
Thus Zn²⁺ does not affect the control of the mRNA level.

BP10 protein localization
Immunolocalization of BP10 protein in prehatching blastulae embryos raised in the presence of Zn²⁺ could not be distinguished from that of the control embryos. The labeling is restricted to the animal two-thirds of the embryo (Ghiglione et al., 1993).

Action of vegetalizing agents
Expression of BP10 transcripts
Embryos were exposed continuously to various concentrations of LiCl ranging from 0 to 90 mM and the the maximal level of mRNA accumulation for each Li⁺ concentration was detected by densitometric scanning of the slot-blots. Li⁺ clearly decreased the amount of mRNA accumulation.

BP10 protein localization
Immunolocalization of BP10 protein in prehatching blastulae of embryos raised in the presence of Li⁺ shows that the size of the territory in which protein is detected is dramatically reduced. This effect is concentration - dependent, as the Li⁺ concentration increases, the size of the territory decreases.
The effect of Li⁺ is time dependent. A maximal effect was obtained when the Li⁺ incubation began within 30 min after fertilization. This effect decreased if Li⁺ was added later, and completely disappeared if lithium was added later than 6 h after fertilization, which is the time when the transcription rate of the gene had nearly reached its peak value in normal embryos (Ghiglione et al., 1993).

Sequences

GenBank:

6003 bp DNA

Regulatory Regions

Regions

Regulatory Connections

Upstream Genes

BP10

Downstream Genes

Evolutionary Homologues

tolloid Drosophila
tolloid-related-1 Drosophila
huBMP-1 human
ASTPR Astacus crayfish
SpAN Strongylocentrotus purpuratus
SuBMP Strongylocentrotus purpuratus

Links

Urchin Web

Bibliography

UrchiNet

Search the GeNet

Stage	Egg	16 cells	Early blastula	Prehatching blastula	Swimming blastula	Mesenchyme blastula	Gastrula	Prism
Level	-	-	-	+	+	+	+	+