Purification,Enzymatic Characterization And Molecular Cloning Of Cathepsin L From Pacific Abalone (Haliotis Discus Hannai)

Pacific abalone （Haliotis discus hanai） is one of the most important economic species ofshellfish. With the increasing of price, the production of Pacific ablone is also growing,especially in Fujian province. However, because of water pollution and disease problems, thereis a growing awareness that pollution may cause catastrophic losses to fisheries farmers. It hasbeen reported that cathepsin L is an important protein involved in immunological regulation,which has much relationship with diseases. But there was no report concerning cathepsin L fromPacific abalone. Therefore, it is necessary to study this proteinase in order to understand thecharacteristic of the enzyme and obtain its full sequence. The results of our present research mayprovide some theories for controlling diseases and give some useful suggestions for abalonebreeding.In this study, cathepsin L was purified to homogeneity from Pacific abalone （Haliotis discushannai） visceral by ammonium sulfate fractionation and column chromatographies includingSP-Sepharose and Sephacryl S-200HR. SDS-PAGE showed that the purified cathepsin Lrevealed two bands with molecular masses of28and28.5kDa, named cathespsin Laand Lb. The28.5kDa band was quite possibly a glycosylated form of the28kDa band. Peptide massfingerprinting revealed that the enzyme from Pacific abalone has a high similarity （80%） in95amino acid residues compared with cathepsin L from pearl oyster （Pinctada fucata）, and66.7%with small abalone （Haliotis diversicolor）. The proteinase revealed optimal activity at35°C andpH5.5. The enzyme activity was effectively inhibited by cystatin proteinases inhibitor E-64, butwas activated by EGTA and EDTA which were the inhibitors for metalloproteinases. Metal ionssuch as Mn²⁺, Cu²⁺，Fe²⁺，Co²⁺restrained the activity of cathepsin L, while Ca²⁺, Mg²⁺couldactivate the enzyme to some degree. Cathepsin L could effectively hydrolyse the substrates ofZ-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA, when the site of P2was hydrophobic amino acids.Using Z-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA as substrates, the kinetic constansts ofcathepsin L was K_m2.34μmol/L and k_cat24.21s^-1，and K_m2.28μmol/L and k_cat16.77s^-1. Tissuedistribution of cathepsin L was investigated by Western blot. It was noticed that cathepsin Lcould positively be detected in ovary and hepatopancreas, while quite less in gill, barely inmuscle, mantle and epipodium. The gene of cathepsin L was cloned based on the information of PMF and the ESTsequence of Haliotis discus discus together with RT-PCR and rapid amplification of cDNA ends（RACE）. The full length of cathepsin L was1148bp with a981bp open reading frame encoding327amino acid residues with a signal peptide of16amino acid residues, and the deducedmolecular weight was36.9kDa and pI was6.89. The sequence named cathepsin L wasregistered in GenBank with accession number of JX569796.1. Compared with the result fromPMF, only2amino acid residues were different, suggesting the cloned gene was quite possiblythe same one corresponing to the purified cathepsin L. Three amino acid residues Cys¹³⁴, His²⁷³and Asn²⁹³ consisting the active site were identified in the sequence of cathepsin L. A potentialglycosylation site with the sequence of N was identified in the primary sequence in residues of217. The secondary and third structure of cathepsin L consists of α-helix, β-sheet and loop.