Biochemical Characterization of a Lysosomal α-Mannosidase from the Starfish Asterias rubens.

Acidic α-mannosidase is an important enzyme and is reported from many different plants and animals. Lysosomal α-mannosidase helps in the catabolism of glycoproteins in the lysosomes thereby playing a major role in cellular homeostasis. In the present study lysosomal α-mannosidase from the gonads of echinoderm Asterias rubens was isolated and purified. The crude protein sample from ammonium sulfate precipitate contained two isoforms of mannosidase as tested by the MAN2B1 antibody, which were separated by anion exchange chromatography. Enzyme with 75 kDa molecular weight was purified and biochemically characterized. Optimum pH of the enzyme was found to be in the range of 4.5-5 and optimum temperature was 37 °C. The activity of the enzyme was inhibited completely by swainsonine but not by 1-deoxymannojirimycin. Ligand blot assays showed that the enzyme can interact with both the lysosomal enzyme sorting receptors indicating the presence of mannose 6-phosphate in the glycan surface of the enzyme. This is the first report of lysosomal α-mannosidase in an active monomeric form. Its interaction with the receptors suggest that the lysosomal enzyme targeting in echinoderms might follow a mannose 6-phosphate mediated pathway similar to that in the vertebrates.

Kinetic and biophysical characterization of a lysosomal α-l-fucosidase from the fresh water mussel, Lamellidens corrianus.

Kinetic and biophysical studies have been carried out on a lysosomal α-l-fucosidase purified from the fresh water mussel, Lamellidens corrianus. The enzyme migrates as a single band in SDS-PAGE as well as native PAGE corresponding to a Mr of 56kDa. Mass spectrometric analysis yielded a molecular mass of 56175.1Da for the enzyme, and peptide mass fingerprinting studies showed that it shares sequence homology with other fucosidases. Zymogram analysis showed that the α-l-fucosidase hydrolyzed 4-methyl umbelliferyl α-l-fucopyranoside. The pH and temperature optima of the enzyme were found to be 5.0-6.0 and 60°C, respectively. The KM, Vmax and kcat values of the enzyme estimated with p-nitrophenyl fucopyranoside are 0.85mM, 27.20 mU/mL and 1.01s-1, respectively. The inhibition constant (Ki) of the enzyme towards l-Fucose is 1.09mM. CD spectral analysis has shown that the protein contains predominantly ß-sheets in its secondary structure. Chemical unfolding studies indicate that α-l-fucosidase unfolds in a broad sigmoidal, cooperative unfolding transition, centered at ∼2.2M for both guanidinium chloride and guanidinium thiocyanate. The present results obtained with the L. corrianus α-l-fucosidase are expected to provide further insights into the various biological processes associated with fucosidases and help in exploiting this enzyme in therapeutic applications.

Jack bean α-mannosidase: amino acid sequencing and N-glycosylation analysis of a valuable glycomics tool.

Jack bean (Canavalia ensiformis) seeds contain several biologically important proteins among which α-mannosidase (EC 3.2.1.24) has been purified, its biochemical properties studied and widely used in glycan analysis. In the present study, we have used the purified enzyme and derived its amino acid sequence covering both the known subunits (molecular mass of ∼66,000 and ∼44,000 Da) hitherto not known in its entirety. Peptide de novo sequencing and structural elucidation of N-glycopeptides obtained either directly from proteolytic digestion or after zwitterionic hydrophilic interaction liquid chromatography solid phase extraction-based separation were performed by use of nanoelectrospray ionization quadrupole time-of-flight mass spectrometry and low-energy collision-induced dissociation experiments. De novo sequencing provided new insights into the disulfide linkage organization, intersection of subunits and complete N-glycan structures along with site specificities. The primary sequence suggests that the enzyme belongs to glycosyl hydrolase family 38 and the N-glycan sequence analysis revealed high-mannose oligosaccharides, which were found to be heterogeneous with varying number of hexoses viz, Man8-9GlcNAc2 and Glc1Man9GlcNAc2 in an evolutionarily conserved N-glycosylation site. This site with two proximal cysteines is present in all the acidic α-mannosidases reported so far in eukaryotes. Further, a truncated paucimannose type was identified to be lacking terminal two mannose, Man1(Xyl)GlcNAc2 (Fuc).

Biochemical characterization of cathepsin D from the mussel Lamellidens corrianus.

A lysosomal cathepsin D (EC 3.4.23.5) was purified to homogeneity from the soft tissues of the fresh water mussel (Lamellidens corrianus) by pepstatin A affinity chromatography. The purified enzyme is a glycoprotein and migrates as a single protein species in native PAGE and shows a single band in SDS-PAGE corresponding to a molecular mass of ~43 kDa. Under both these conditions cathepsin D hydrolyzes hemoglobin as shown by zymogram analysis. The purified enzyme cross-reacts with an antiserum to purified starfish (Asterias rubens) cathepsin D. Additionally, the enzyme was recognized by the starfish lysosomal enzyme targeting receptors (mannose 6-phosphate receptors: MPR 300 and 46) in ligand blot analysis. The KM value of the purified enzyme with hemoglobin is 1.5mM. pH and temperature optimum for the enzyme are 3.5 and 60 °C respectively.

Isolation, affinity purification and biochemical characterization of a lysosomal cathepsin D from the deuterostome Asterias rubens.

Cathepsin D (EC 3.4.23.5) is one of the lysosomal enzymes responsible for proteolytic degradation in cells. By virtue of its mannose 6-phosphate residues, shortly after its synthesis, it is recognized by the receptors in the trans-Golgi network that mediate its transport to the lysosomes. The mammalian enzyme has been extensively characterized and several forms of cathepsin have also been identified. Cathepsins have also been isolated from other vertebrates and invertebrates and recent studies suggest that the lysosomal sorting machinery is evolutionarily conserved from fish to mammals. We recently characterized the putative mannose 6-phosphate receptors from the invertebrate starfish (Asterias rubens). In the present study we affinity purified the cathepsin D from this animal and biochemically characterized the same. Purified enzyme migrated as a single band on SDS-PAGE corresponding to a molecular mass of 45 kDa. The protein bound specifically to Con A-Sepharose gel and is glycosylated. The deglycosylated enzyme showed a molecular mass of ~40 kDa. Furthermore, an antibody raised for the purified enzyme in a rabbit recognizes the crude, the purified enzyme as well as the deglycosylated product in a western blot experiment. The enzyme in the extracts of different tissues can also be quantified by ELISA. We have further evaluated the binding of purified starfish cathepsin D with its receptor, MPR 300 (mannose 6-phosphate receptor) by immunoprecipitation. Cross-linking experiments using purified cathepsin D and MPR 300 revealed a cross-linked product that migrated with a higher molecular mass (345 kDa) compared to the enzyme (45 kDa). Furthermore the specificity of this interaction was also tested in a ligand blot experiment.

Reptilian MPR 300 is also the IGF-IIR: cloning, sequencing and functional characterization of the IGF-II binding domain.

The mammalian cation-independent mannose 6-phosphate/insulin-like growth factor (IGF)-II receptor binds IGF-II with high affinity. Ligands transported by the MPR 300/IGF-IIR include IGF-II and mannose 6-phosphate-modified proteins. By targeting IGF-II to lysosomal degradation, it plays a key role in the maintenance of correct IGF-II levels in the circulation and in target tissues. Although, from our studies we found homologous receptor in calotes but its functional significance was not known. We present here the first report on the calotes MPR 300/IGF-IIR binds IGF-II with K(d) of 12.02 nM; these findings provide new and strong evidence that MPR 300/IGF-IIR in Calotes versicolor binds IGFII with high affinity.