Given that synthesis and maturation of CD requires >1.5 h [42], the above finding could be explained assuming that CD is not synthesized in UFE, while at 30% epiboly (4.66 hpf) the amount of CD protein accumulated is still under the detection limit of the western blotting. Coomassie blue staining of the protein loaded on gel shows a prominent band at high molecular weight in the homogenates Afatinib manufacturer of UFE and 30% epiboly (Fig. 2A, lower panel). This band likely contains vitellogenin, since it is missing in samples from 1 to 4 dpf that had been de-yolked prior to homogenization. Figure 2 Zebrafish cathepsin D protein expression. The polyclonal antibody produced in our laboratory specifically recognizes rodent and human CD [43]. We tested its ability to also recognize zebrafish CD in transfected human neuroblastoma SH-SY5Y cells over-expressing transgenic zebrafish CD.

To this end, we performed a western blotting experiment in which the homogenates of transfected SH-SY5Y cells over-expressing human CD, of zebrafish embryonic fibroblast PAC2 cells and of 4 dpf larvae were also included as controls. In accord with our previous report [43], the antibody recognized the human CD forms (the precursor and the mature large chain) of expected size (Fig. 2C). The antibody also detected a band of 41 kDa, that corresponds to the predicted size of mature zebrafish CD, in homogenates of zebrafish larva, of PAC2 cells and of zebrafish cDNA-transfected SH-SY5Y cells. In the latter sample, a band of 43 kDa, that corresponds to the expected size of zebrafish pro-CD, was also detected (Fig. 2C).

It is to note that the precursor of CD (either human and zebrafish) is detectable only in transfected over-expressing cells. Also, it is remarkable that contrary to human CD, that completes its maturation becoming a double-chain, zebrafish CD remains (mainly) as a single-chain protein. This is a common feature of fish CD [44], [45]. To further confirm the predicted molecular weight of mature zebrafish CD, we performed a purification of its active isoform by affinity chromatography using a pepstatinyl-agarose column (Fig. 3). The binding of CD to Pepstatin A occurs through the active site [46] and strictly depends on the proper folding of the mature polypeptide [41], [47]. The purified fractions were resolved by SDS-PAGE and the proteins transferred onto nitrocellulose filter were revealed with our rabbit polyclonal anti-CD antiserum [40], [41].

The experiment confirms that the molecular weight of the mature and enzymatically active zebrafish CD is of 41 kDa, Batimastat as predicted by its cDNA, and that the protease exists as a single-chain isoform, in agreement with the above data. This molecular form of zebrafish CD was shown active toward several substrates at acid pH and in a Pepstatin A-sensitive manner, as revealed by enzyme assay using PAC2 cell homogenate as source (data not shown).