Identification and characterization of a cathepsin D homologue from lampreys (Lampetra japonica).

Cathepsin D (EC 3.4.23.5) is a lysosomal aspartic proteinase of the pepsin superfamily which participates in various digestive processes within the cell. In the present study, the full length cDNA of a novel cathepsin D homologue was cloned from the buccal glands of lampreys (Lampetra japonica) for the first time, including a 124-bp 5' terminal untranslated region (5'-UTR), a 1194-bp open reading frame encoding 397 amino acids, and a 472-bp 3'-UTR. Lamprey cathepsin D is composed of a signal peptide (Met 1-Ala 20), a propeptide domain (Leu 21-Ala 48) and a mature domain (Glu 76-Val 397), and has a conserved bilobal structure. Cathepsin D was widely distributed in the buccal glands, immune bodies, hearts, intestines, kidneys, livers, and gills of lampreys. After challenging with Escherichia coli or Staphylococcus aureus, the expression level of lamprey cathepsin D in the buccal gland was 8.5-fold or 6.5-fold higher than that in the PBS group. In addition, lamprey cathepsin D stimulated with Escherichia coli was also up-regulated in the hearts, kidneys, and intestines. As for the Staphylococcus aureus challenged group, the expression level of lamprey cathepsin D was found increased in the intestines. The above results revealed that lamprey cathepsin D may play key roles in immune response to exogenous pathogen and could serve as a potential antibacterial agent in the near future. In addition, lamprey cathepsin D was subcloned into pcDNA 3.1 vector and expressed in the human embryonic kidney 293 cells. The recombinant lamprey cathepsin D could degrade hemoglobin, fibrinogen, and serum albumin which are the major components in the blood, suggested that lamprey cathepsin D may also act as a digestive enzyme during the adaptation to a blood-feeding lifestyle.

Isolation, biochemical characterization, and molecular modeling of American lobster digestive cathepsin D1.

An aspartic proteinase was isolated from American lobster gastric fluid. The purified cathepsin D runs as a single band on native-PAGE displaying proteolytic activity on a zymogram at pH 3.0, with an isoelectric point of 4.7. Appearance of the protein in SDS-PAGE, depended on the conditions of the gel electrophoresis. SDS treatment by itself was not able to fully unfold the protein. Thus, in SDS-PAGE the protein appeared to be heterogeneous. A few minute of boiling the sample in the presence of SDS was necessary to fully denature the protein that then run in the gel as a single band of ~50 kDa. The protein sequence of lobster cathepsin D1, as deduced from its mRNA sequence, lacks a 'polyproline loop' and ß-hairpin, which are characteristic of some of its structural homologues. A comparison of amino acid sequences of digestive and non-digestive cathepsin D-like enzymes from invertebrates showed that most cathepsin D enzymes involved in food digestion, lack the polyproline loop, whereas all non-digestive cathepsin Ds, including the American lobster cathepsin D2 paralog, contain the polyproline loop. We propose that the absence or presence of this loop may be characteristic of digestive and non-digestive aspartic proteinases, respectively.

Cathepsin D-like aspartic proteinase occurring in a maize weevil, Sitophilus zeamais, as a candidate digestive enzyme.

The maize weevil, Sitophilus zeamais, is an insect pest infesting rice and corn seeds. We identified an aspartic proteinase (AP) digesting rice glutelin in the alimentary tract of S. zeamais. The mRNA encoding the AP (SAP1) was expressed in the larvae foregut and in the adult midgut. These results indicate that SAP1 is probably digestive enzyme of S. zeamais.