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Three novel cDNAs encoding serine proteases, that may play a role in early vertebrate development, have been identified from Xenopus laevis. These Xenopus cDNAs encode trypsin-like serine proteases and are designated Xenopus embryonic serine protease (Xesp)-1, Xesp-2, and XMT-SP1, a homolog of human MT-SP1. Xesp-1 is likely to be a secreted protein that functions in the extracellular space. Xesp-2 and XMP-SP1 are likely to be type II membrane proteases with multidomain structures. Xesp-2 has eight low density lipoprotein receptor (LDLR) domains and one scavenger receptor cysteine-rich (SRCR) domain, and XMT-SP1 has four LDLR domains and two CUB domains. The temporal expressions of these serine protease genes show distinct and characteristic patterns during embryogenesis, and they are differently distributed in adult tissues. Overexpression of Xesp-1 caused no significant defect in embryonic development, but overexpression of Xesp-2 or XMT-SP1 caused defective gastrulation or apoptosis, respectively. These results suggest that these proteases may play important roles during early Xenopus development, such as regulation of cell movement in gastrulae.
Fig. 1.
Predicted domain structure of Xesp-1, Xesp-2 and XMT-SP1. Xesp-1 has a signal peptide and a serine protease domain. Xesp-2 has a transmembrane (TM) binding domain, eight LDLR domains, a SRCR domain, and a serine protease domain. XMT-SP1 has a transmembrane (TM) binding domain, two CUB domains, four LDLR domains, and a serine protease domain.
Fig. 2.
Multiple sequence alignments of the structural motifs. (A) Alignment of the serine protease domains of Xesp-1, Xesp-2, and XMT-SP1 with Epithin, MT-SP1, and Trypsin. The catalytic triad residues (His, Asp, Ser) are in black. The residues of the substrate binding pocket are in gray. (B) Alignment of the LDL receptor domains of Xesp-2 and XMT-SP1 with MT-SP1. (C) Alignment of the CUB domains of XMT-SP1 with oviductin. (D) Alignment of the SRCR domain of Xesp-2 with TMPRSS2 and mouse macrophage scavenger receptor type I. (AâD) The boxes indicate the conserved Cys residues.
Fig. 3.
The mRNA expression patterns of Xesp-1, Xesp-2, and XMT-SP1 in embryonic development (A) and adult tissues (B) analyzed by RTâPCR. ODC and EF1-α were used as internal controls.
Fig. 4.
Effect of ectopic expression of Xesp-2 and XMT-SP1. The wild-type (A) or mutant (B) mRNA encoding Xesp-2 was injected into both blastomeres at the two-cell stage. Overexpression of the wild-type Xesp-2 disturbed the closure of blastopore, but there was no defect after injection of the mutant form. Unilateral injection of wild-type XMT-SP1 at the two-cell stage results in cell death in the injected side (C) where non-adherent cells are observed (D). TUNEL assay also shows apoptotic cell death in the side injected with wild-type XMT-SP1 (E), but no apoptotic signal after injection of the mutant form (F).
