Sains Malaysiana 43(8)(2014): 1139–1148

Molecular Characterisation and Expression Analysis of Cathepsin D

from the Asian Seabass Lates calcarifer

(Pencirian Molekul dan Analisis Pengekspresan Katepsin D daripada Ikan Siakap Lates calcarifer)

SHARIZA AZIZAN¹, KIEW-LIAN WAN^1,2* & ADURA MOHD-ADNAN²

¹School of Biosciences and Biotechnology, Faculty of Science and Technology

Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor D.E. Malaysia

²Malaysia Genome Institute, Jalan Bangi, 43000 Kajang, Selangor, D.E. Malaysia

Received: 18 May 2013/Accepted: 26 November 2013

ABSTRACT

The lysosomal aspartic proteinase cathepsin D is an acute phase protein involved in various physiological processes, including vitellogenesis, yolk processing and immune responses. In this study, we characterised the cathepsin D from the Asian seabass Lates calcarifer and examined its expression profile during infection. The complete coding sequence of L. calcarifer cathepsin D consists of 1191 nucleotides, encoding a 396 amino acid protein molecule that is made up of a putative signal peptide, a leader peptide and a mature peptide. Phylogenetic analyses showed that two types of cathepsin D are present in the teleost lineage i.e. cathepsin D1 and D2, whereas higher vertebrates possess only one type of cathepsin D. L. calcarifer cathepsin D was clustered together with cathepsin D1 from other teleosts. Compared to mammalian sequences, L. calcarifer cathepsin D lacks the β-hairpin loop that forms the double chain and is present as a single chain peptide with conserved aspartic active sites like other fish. Both multiple sequence alignment and phylogenetic analysis indicated that the L. calcarifer cathepsin D sequence codes for cathepsin D1 and suggested that it shares the same functions with cathepsin D from other fish. Expression profiling analysis of cathepsin D in L. calcarifer infected with Aeromonas hydrophila showed that it is up-regulated in immune-related tissues such as gills, spleen and liver, suggesting that cathepsin D plays an important role in the innate immune response of L. calcarifer against pathogens.

Keywords: Acute phase protein; aspartic proteinase; expression profile; innate immune response

ABSTRAK

Katepsin D, sejenis proteinase aspartik lisosom merupakan protein fasa akut yang terlibat dalam pelbagai proses fisiologi, termasuk vitelogenesis, pemprosesan yolka dan gerak balas keimunan. Dalam kajian ini, kami telah mencirikan katepsin D daripada ikan siakapLates calcarifer dan mengkaji profil pengekspresannya semasa infeksi. Jujukan pengekodan lengkap katepsin D L. calcarifer terdiri daripada 1191 nukleotida yang mengekod molekul protein bersaiz 396 asid amino yang merangkumi satu peptida isyarat putatif, satu peptida mendahului dan satu peptida matang. Analisis filogenetik menunjukkan bahawa terdapat dua jenis katepsin D hadir dalam susur galur teleost iaitu katepsin D1 dan D2, sementara vertebrata peringkat tinggi hanya mempunyai satu jenis katepsin D. Katepsin D L. calcarifer dikelompokkan bersama dengan katepsin D1 ikan teleost lain. Berbanding dengan jujukan mamalia, katepsin D L. calcarifer didapati tidak mempunyai jujukan gelung pin rambut β yang membentuk rantai ganda dua dan wujud sebagai rantai tunggal peptida dengan kehadiran tapak aktif aspartik terpulihara seperti ikan lain. Penjajaran jujukan berbilang dan analisis filogenetik menunjukkan bahawa jujukan katepsin D L. calcarifer mengekodkan katepsin D1 dan mencadangkan ia mempunyai fungsi yang sama dengan katepsin D ikan lain. Analisis profil pengekspresan katepsin D dalamL. calcarifer terinfeksi Aeromonas hydrophila mendedahkan bahawa pengekspresannya meningkat dalam tisu berkait-keimunan seperti insang, limpa dan hepar mencadangkan bahawa katepsin D memainkan peranan yang penting dalam gerak balas keimunan semula jadiL. calcarifer terhadap patogen.

Kata kunci: Gerak balas keimunan semula jadi; profil pengeskpresan; proteinase aspartik; protein fasa akut

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*Corresponding author; email: klwan@ukm.edu.my