Biomedical subjects

Md Nazmul Ahsan

Publications and source records attributed to Md Nazmul Ahsan.

5 recordsLinked to original sources

PubMed2006

Characterization of cDNA clones encoding two distinct cathepsins with restricted expression pattern in a marine pelagic fish.

Cathepsin L (EC 3.4.22.15) from aquatic animals are quite stable and active at neutral or alkaline pH values while their mammalian equivalents work at an acidic environment of the lysosomes. To understand the molecular properties at the gene level we employed a PCR-based strategy using degenerate oligonucleotide primers to isolate cathepsin L-like genes from anchovy Engraulis japonicus. As a result, we obtained two closely related genes encoding cathepsins (aCat1 and aCat2) similar to both cathepsins L and S from other organisms. The predicted precursor protein of 324 amino acid residues for genes differed in six residues and contained conserved residues characteristic of cathepsin L-like cysteine proteases. Phylogenetic analyses failed to produce any precise relationships of aCat1 and aCat2 with other cysteine proteases. However, with a bootstrap value less than 50, these two fish cathepsins formed a separate group to that bearing cathepsins L and S of various organisms. Interestingly, unlike mammalian cathepsin L transcripts of aCat1 and aCat2 were almost exclusively detected in the stomach suggesting that the fish homologues are non-lysosomal secretory enzymes present in the extracellular acidic environment of the stomach and that marine teleosts developed digestive cysteine proteases as a result of evolutionary pressure in response to varying dietary conditions.

Amino Acid Sequence↗

PubMed2003

Purification and characterization of collagenolytic proteases from the hepatopancreas of northern shrimp (Pandalus eous).

Three gelatinolytic proteases (A1, A2, and B) were purified using a synthetic substrate, DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, from the hepatopancreas of Northern shrimp (Pandalus eous) by several chromatographic steps involving hydroxyapatite column chromatography, gel filtration on Superdex75, and ion-exchange chromatography on a MonoQ column. Collagenolytic proteases A2 and B, but not protease A1, were demonstrated to digest native porcine type I collagen at 25 degrees C and pH 7.5. Further characterizations of these two collagenolytic proteases showed that the pH optimum of enzyme A2 against DNP-peptide was found to be 11, whereas that of enzyme B was 8.5. The optimum temperature ranged between 40 and 45 degrees C for both enzymes, although enzyme B appeared to be thermally more stable than enzyme A2 at pH 7.5. Both enzymes were strongly inhibited by PMSF and antipain, which suggests that they belong to collagenolytic serine proteases.

Animals↗

PubMed2003

Heterologous expression in Pichia pastoris and single-step purification of a cysteine proteinase from northern shrimp.

A distinct cysteine proteinase (NsCys) of northern shrimp Pandalus borealis belonging to cathepsin L subgroup of the papain superfamily has been overexpressed as a precursor form (proNsCys) in Pichia pastoris. We adopted a simple and quick procedure to generate an expression cassette by constructing a donor vector harboring proNsCys followed by recombination with an acceptor vector in a way so that the proNsCys gene was placed downstream of the methanol-inducible AOX1 promoter and alpha-mating factor signal sequence gene. In addition, we used glycerol complex medium that supported high growth of yeast before induction while induction was carried out in minimal methanol medium thereby facilitating the secreted protein to be purified with a single size-exclusion chromatography. The recombinant enzyme was purified in two enzymatically active fractions: both corresponding to mature NsCys with, however, the major one comprising two molecular species of NsCys which had their severed prodomain non-covalently attached. The overall yield was about 100 mg of crude or 60 mg of purified recombinant enzyme comprising both mature and prodomain-attached forms of NsCys per liter of yeast culture. The recombinant NsCys was biologically active as observed by gelatin zymography and its ability to cleave Z-Phe-Arg-MCA, a synthetic substrate for cathepsin L. The development of the system reported here provides a cost-effective and easy to manipulate expression system to obtain large quantities of fully functional shrimp enzyme that will enable the functional characterization of this unique enzyme for both research and industrial purposes.

Amino Acid Sequence↗

PubMed2003

Molecular cloning and characterization of cathepsin B from the hepatopancreas of northern shrimp Pandalus borealis.

We cloned a cDNA encoding cathepsin B from the hepatopancreas of northern shrimp Pandalus borealis (NsCtB). Nucleotide sequence of the isolated clone encoded a preproenzyme of 328 amino acids, comprising a 15-residue putative signal peptide, a 60-residue propeptide and the 253-residue mature enzyme. The mature NsCtB was 53% identical to human cathepsin B and conserved all the structural features characteristic of cysteine protease. The presence of an occluding loop in the mature region, a unique feature of cathepsin B, suggested the shrimp protein to be cathepsin B. Northern blot analysis revealed expression of NsCtB transcripts exclusively in the hepatopancreas tissues, suggesting a possible digestive role of this enzyme. An interesting feature of NsCtB was its remarkably high negative charge in comparison with other cysteine proteases, which was predicted to effectively locate and guide the positively charged residues of a substrate into the binding cleft. We also observed a repertoire of cysteine protease activities in the acidic milieu of shrimp hepatopancreas using synthetic substrates specific to various cathepsins. The activity profile revealed cathepsin B as the single most dominant enzyme with a specific activity comparable to that attributable to combined activities of other cathepsins. This activity could be blocked by E-64, a cysteine protease inhibitor, but not by Z-Phe-Tyr (t-Bu)-CHN(2), a specific inhibitor of cathepsin L.

Animals↗

PubMed2003

Molecular cloning and functional characterization of crustapain: a distinct cysteine proteinase with unique substrate specificity from northern shrimp Pandalus borealis.

A cDNA clone encoding a cysteine proteinase of the papain superfamily has been isolated from the hepatopancreas of northern shrimp Pandalus borealis (NsCys). NsCys shares the highest identity of 64% with a cathepsin L-like cysteine proteinase from lobster, and its identity to the well-characterized mammalian cathepsins S, L, and K falls within a narrow range of 54-59%. However, it differs from each of these cathepsins in certain key residues including, for example, the unique occurrence of tryptophan and cysteine residues at the structurally important S2 subsite. Consequently, NsCys produced in Pichia pastoris appears to be distinct in various physicokinetic properties. The recombinant enzyme is active and stable over a wide range of pH values, and its substrate specificity is unusual, as demonstrated by its poor affinity for phenylalanine residues. Instead, it shows the highest specificity for proline residues, a property similar to cathepsin K. Unlike cathepsin K, however, NsCys cleaves valine residues more efficiently than leucine. Similar results were obtained with the natural peptide substrate glucagon. The shrimp proteinase is further distinguished by its potent collagenolytic activity, resulting in a cleavage pattern reminiscent of bacterial collagenase. To distinguish such unique structural and enzymatic properties, we propose the trivial name "crustapain" for the shrimp proteinase, indicating that it is a papain-like cysteine proteinase from a crustacean species.

Amino Acid Sequence↗