News from an Ancient World: Two Novel Astacin Metalloproteases from the Horseshoe Crab
In this work, we report the cloning, heterologous expression, and characterization of two novel astacin proteases from the chelicerate Limulus polyphemus (horseshoe crab), designated as LAST (Limulus astacin) and LAST_MAM (Limulus astacin containing a MAM domain), respectively. The expression pattern showed ubiquitous occurrence of LAST_MAM, while LAST was predominantly restricted to the eyes and brain, indicating a function in the nervous system. Both enzymes contain the characteristic metzincin-type zinc-binding region and Met turn. While LAST is made up only of the typical prodomain and astacin-like protease domain, LAST_MAM contains an additional MAM (meprin A5 protein tyrosine phosphat…
Proteomic Analyses Reveal an Acidic Prime Side Specificity for the Astacin Metalloprotease Family Reflected by Physiological Substrates
Astacins are secreted and membrane-bound metalloproteases with clear associations to many important pathological and physiological processes. Yet with only a few substrates described their biological roles are enigmatic. Moreover, the lack of knowledge of astacin cleavage site specificities hampers assay and drug development. Using PICS (proteomic identification of protease cleavage site specificity) and TAILS (terminal amine isotopic labeling of substrates) degradomics approaches >3000 cleavage sites were proteomically identified for five different astacins. Such broad coverage enables family-wide determination of specificities N- and C-terminal to the scissile peptide bond. Remarkably, me…
Two α subunits and one β subunit of meprin zinc-endopeptidases are differentially expressed in the zebrafish Danio rerio
Abstract Meprins are members of the astacin family of metalloproteases expressed in epithelial tissues, intestinal leukocytes and certain cancer cells. In mammals, there are two homologous subunits, which form complex glycosylated disulfide-bonded homo- and heterooligomers. Both human meprin α and meprin β cleave several basement membrane components, suggesting a role in epithelial differentiation and cell migration. There is also evidence that meprin β is involved in immune defence owing to its capability of activating interleukin-1β and the diminished mobility of intestinal leukocytes in meprin β-knockout mice. Here we show for the first time by reverse transcription PCR, immunoblotting a…
Let it flow: Morpholino knockdown in zebrafish embryos reveals a pro-angiogenic effect of the metalloprotease meprin alpha2.
BACKGROUND: Meprin metalloproteases are thought to be involved in basic physiological functions such as cell proliferation and tissue differentiation. However, the specific functions of these enzymes are still ambiguous, although a variety of growth factors and structural proteins have been identified as meprin substrates. The discovery of meprins alpha(1), alpha(2) and beta in teleost fish provided the basis for uncovering their physiological functions by gene silencing in vivo. METHODOLOGY/PRINCIPAL FINDINGS: A Morpholino knockdown in zebrafish embryos targeting meprin alpha(1) and beta mRNA caused defects in general tissue differentiation. But meprin alpha(2) morphants were affected more…