Danar Praseptiangga

Danar Praseptiangga, S.TP., M.Sc., Ph.D. 

Lahir di Surakarta, 9 September 1981. Pria yang memiliki NIP 198109092005011002 adalah staf Pengajar pada Fakultas Pertanian UNS. Riwayat pendidikan tinggi yang berhasil ditempuh adalah tahun 2003 lulus sarjana (S-1) dari Universitas Gadjah Mada  pada bidang ilmu: Teknologi Pangan dan Hasil Pertanian, tahun 2009 berhasil menyelesaikan master (S-2) dari Hiroshima University, Japan pada bidang ilmu: Food Science and Biotechnology, dan pada tahun 2012 telah berhasil menyelesaikan program Doktor (S-3) dari Hiroshima University, Japan untuk bidang ilmu: Food Science and Biotechnology. Judul dan ringkasan disertasi disajikan dalam Bahasa English sebagai berikut.

Many algal lectins share some common characteristics of low-molecular weight, monomeric forms, thermostability, divalent cation-independent hemagglutination, and having affinity only for glycoproteins but not for monosaccharides. These properties of algal lectins are dissimilar to most of land plant lectins that have affinity for monosaccharides and consist of oligomeric forms. Recently, however, monosaccharide-binding lectins have been reported from  several  species  of  green  algae,  especially  from  the  genus  Codium,  including  two subspecies of C. fragile, C. tomentosum, and C. giraffa. They are commonly specific for N-acetylgalactosamine (GalNAc) and consist of oligomeric forms, except a monomeric lectin from  C.  giraffa.  The  resemblance  of  lectin  properties  may  be  derived  from  the  close evolutionary distance between the  green algae of the genus Codium and the land plant, although there is no information on the primary structures of Codium lectins. Some Codium lectin shows preferential affinity for a carbohydrate marker of cancer. Thus, the lectins of the genus Codium are interesting targets for their application as biochemical and clinical reagents, as well as to provide insight into the molecular evolution of lectins in the plant kingdom. This study was performed to clarify the biochemical properties of the lectins from three other species of the genus Codium, including C. barbatum, C. pugniformis, and C. latum.

The C. barbatum lectin (CBA) was purified by conventional purification methods for proteins.  The  hemagglutination of CBA was not inhibited by GalNAc and the other monosaccharides  examined.  However,  it  was  inhibited  by complex-type N-glycan-linked glycoproteins. In the binding assay with 26 pyridylaminated (PA-) oligosaccharides using a centrifugal ultrafiltration-HPLC method, CBA exclusively bound to core (α1-6) fucosylated complex type N-glycans and did not bind to the other oligosaccharides examined, suggesting its strict specificity for core (α1-6) fucose. The primary structure of CBA was determined by a combination of ESI-MS, Edman degradation, and cDNA cloning. CBA consisted of an SS-linked homodimer of a 9257-Da polypeptide containing seven cysteine residues, all of which  were  involved  in  disulfide  linkages.  The  cDNA  of  the  CBA  subunit  coded  a polypeptide (105 amino acids)  including the signal peptide of 17 residues. The calculated molecular mass from the deduced sequence was 9705 Da, implying that the four C-terminal amino acids of the CBA proprotein subunit were post-translationally truncated to afford the mature subunit (84 amino acids). No significantly similar sequences were found during an in silico search, indicating CBA to be a novel protein. CBA is the first Codium lectin  whose primary structure has been elucidated.

The C.  pugniformis  lectin  (CPA)  was  purified  by  a  combination  of  ammonium sulfate-precipitation and affinity  chromatography on a bovine submaxillary mucin-immobilized column. The lectin was adsorbed to the affinity column and specifically eluted  with  0.2  M  GalNAc.  From  SDS-PAGE,  gel  filtration,  and  N-terminal  sequence analysis of the purified lectin, CPA was concluded to be a non-covalently linked oligomer of two distinct polypeptides of 9.5 kDa and 8.5 kDa. The hemagglutination activity of CPA was inhibited by GalNAc, but not by the other monosaccharides  examined, like other known Codium lectins. In the binding assay with 26 PA-oligosaccharides, however, CPA exclusively bound to the oligosaccharides bearing non-reducing terminal β-GalNAc residue and did not to the  other oligosaccharides. Thus CPA belongs to a new lectin group that is specific for β-GalNAc.

The C. latum lectin (CLA) was purified by conventional purification methods for proteins.  The  hemagglutination  activity  of  CLA was  not  inhibited  by  monosaccharides including GalNAc. It was  inhibited only by porcine thyroglobulin and its asialo-derivative among  glycoproteins  examined.  In  oligosaccharide-binding assay, however, CLA has  no significant affinity for 26 PA-oligosaccharides  examined, suggesting its strict and unique carbohydrate-binding  property.  The  primary  structure  of  CLA   was  determined  by  a combination of ESI-MS, Edman degradation, and cDNA cloning. CLA was a  monomeric protein of 15864 Da and the CLA cDNA coded a polypeptide of 169 amino acids, including the signal peptide of 20 residues. The calculated molecular mass (15870 Da) of the deduced amino acid sequence coincided well with the determined mass by ESI-MS. CLA had three repeat regions of similar sequences  and six cysteine residues involved in three intrachain disulfide linkages. CLA shows no significant sequence similarity with other known lectins, including  CBA.  However,  in  silico  search  revealed  that  CLA  belongs  to  the  ‘fascin’ superfamily having a beta-trefoil topology with internal three repeat regions, which have also been reported for carbohydrate-recognition domains of some land plant lectins.

In this study, three lectins were newly isolated and characterized from three species of the genus Codium. Their biochemical properties were obviously different from each other, and only CPA resembled other known Codium lectins in having an oligomeric structure and binding affinity  for GalNAc. Thus, there seems to be diverse functions and structures of lectins within the genus Codium.  The oligosaccharide-binding specificities of CBA, CPA, and CLA were unique, promising their uses as  valuable reagents to decode carbohydrate structures.

Keywords: Codium barbatum, Codium pugniformis, Codium latum, lectins, carbohydrate-binding specificity, cDNA cloning, primary structure