Purification and biochemical/biophysical characterization of two hexosaminidases from the fresh water mussel, Lamellidens corrianus.

Two thermostable isoforms of a hexosaminidase were purified to homogeneity from the soluble extract of fresh water mussel Lamellidens corrianus, employing a variety of chromatographic techniques. Hexosaminidase A (HexA) is a heterodimer with subunit masses of ~80 and 55 kDa. Hexosaminidase B (HexB) is a homodimer with a subunit mass of 55-60 kDa. Circular dichroism spectroscopic studies indicated that both HexA and HexB contain ß-sheet as the major secondary structural component with considerably lower content of α-helix. The temperature and pH optima of both the isoforms were found to be 60 °C and 4.0, respectively. The IC50 values for HexA with N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, d-galactosamine, d-glucosamine, methyl α-d-mannopyranoside and d-mannose are 3.7, 72.8, 307, 216, 244 and 128 mM, respectively, whereas the corresponding IC50 values for HexB were estimated as 5.1, 61, 68, 190, 92 and 133 mM, respectively. Kinetic parameters KM and Vmax for HexA and B with p-nitrophenyl N-acetyl-ß-d-glucosaminide are 4 mM, 0.23 µmol·min-1·mL-1 and 2.86 mM, 0.29 µmol·min-1·mL-1, respectively, and with p-nitrophenyl N-acetyl-ß-d-galactosaminide are 4.5 mM, 0.054 µmol·min-1·mL-1 and 1.4 mM, 0.14 µmol·min-1·mL-1, respectively. GalNAc inhibited both isoforms in a non-competitive manner, whereas a mixed mode of inhibition was observed with GlcNAc with both forms.

Lysosomal enzymes and mannose 6-phosphate receptors in the lacrimal drainage system: Evidence and its potential implications.

PURPOSE: To investigate the presence and patterns of lysosomal enzymes and mannose 6-phosophate receptor (MPRs) in human lacrimal drainage system. METHODS: The study was performed on healthy lacrimal sacs and nasolacrimal ducts obtained from exenteration samples immediately after surgery and frozen at -80°C for subsequent analysis. Soluble proteins' extract was used for enzyme assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), native PAGE, activity staining, and western blot analysis. Membrane proteins were separately assessed for detection of mannose 6-phosphate receptors, MPR 46. Sepharose gels, 4-methylumbelliferyl substrates, and antibodies against common lysosomal enzymes and MPRs were used. Enzyme assays were carried out in triplicate to ascertain the results. RESULTS: Differential lysosomal enzyme activities were documented, and among them acid phosphatase and ß-hexosaminidase were found to be high. Western blot analysis using enzyme antibodies and subsequent activity staining confirmed strong signals for moderately expressed enzymes such as fucosidase, glucuronidase, and mannosidase. Membrane extracts demonstrated the presence of MPR 46, which indicates the possible roles of cation-dependent MPRs in lysosomal targeting in human lacrimal drainage system. CONCLUSION: This study provides a proof of principle for the presence of differential lysosomal activity and mannose 6-phosphate ligand transport receptors in human lacrimal drainage system and hypothesizes the potential implications of their dysfunctions.

Expression of Surfactant Proteins in the Human Canaliculus: Evidence and Potential Insights Into the Tear Flow Dynamics.

PURPOSE: To investigate the presence and distribution patterns of 6 surfactant proteins (SPs) in the human lacrimal canaliculus. METHODS: The study was performed on fresh frozen cadaveric samples of canaliculi. Immunohistochemical labeling was performed for assessing the presence and distribution of SP: SP-A, SP-B, SP-C, SP-D, SP-G/SFTA2, and SP-H/SFTA3. Immunofluorescence double staining was performed using the respective fluorescein-conjugated antibodies and the results were scored as positive or negative and the distribution pattern within the canalicular system was assessed. Western blot analysis was performed on the protein content which was resolved by reducing 15% sodium dodecyl sulfate-polyacrylamide electrophoresis and bands were studied following staining with primary and secondary antibodies. Human lung tissues were used as controls. RESULTS: Fluorescence double staining with 4,6-diamidino 2-pheynlindole and SPs showed strong immunostaining for SP-A, SP-B, SP-C, SP-D, and SP-H/SFTA3. The positive immunofluorescence was noticed across all the layers of the epithelium but not the subepithelial structures. The expression was noted on the surfaces and superficial cytoplasm of the superficial and deep epithelial cells. There was no expression of SP-G/SFTA2 across the canalicular system. Western blot analysis of the proteins confirmed and concurred with the immunofluorescence findings. CONCLUSIONS: This study provides a proof of principle for the presence of SPs known from lungs in the canalicular system and hypothesizes their possible functions and also their potential role in the tear flow dynamics between the ocular surface and the lacrimal drainage system.

Lysosomal enzymes and their receptors in invertebrates: an evolutionary perspective.

Lysosomal biogenesis is an important process in eukaryotic cells to maintain cellular homeostasis. The key components that are involved in the biogenesis such as the lysosomal enzymes, their modifications and the mannose 6-phosphate receptors have been well studied and their evolutionary conservation across mammalian and non-mammalian vertebrates is clearly established. Invertebrate lysosomal biogenesis pathway on the other hand is not well studied. Although, details on mannose 6-phosphate receptors and enzymes involved in lysosomal enzyme modifications were reported earlier, a clear cut pathway has not been established. Recent research on the invertebrate species involving biogenesis of lysosomal enzymes suggests a possible conserved pathway in invertebrates. This review presents certain observations based on these processes that include biochemical, immunological and functional studies. Major conclusions include conservation of MPR-dependent pathway in higher invertebrates and recent evidence suggests that MPR-independent pathway might have been more prominent among lower invertebrates. The possible components of MPR-independent pathway that may play a role in lysosomal enzyme targeting are also discussed here.

The novel Drosophila lysosomal enzyme receptor protein mediates lysosomal sorting in mammalian cells and binds mammalian and Drosophila GGA adaptors.

Biogenesis of lysosomes depends in mammalian cells on the specific recognition and targeting of mannose 6-phosphate-containing lysosomal enzymes by two mannose 6-phosphate receptors (MPR46, MPR300), key components of the extensively studied receptor-mediated lysosomal sorting system in complex metazoans. In contrast, the biogenesis of lysosomes is poorly investigated in the less complex metazoan Drosophila melanogaster. We identified the novel type I transmembrane protein lysosomal enzyme receptor protein (LERP) with partial homology to the mammalian MPR300 encoded by Drosophila gene CG31072. LERP contains 5 lumenal repeats that share homology to the 15 lumenal repeats found in all identified MPR300. Four of the repeats display the P-lectin type pattern of conserved cysteine residues. However, the arginine residues identified to be essential for mannose 6-phosphate binding are not conserved. The recombinant LERP protein was expressed in mammalian cells and displayed an intracellular localization pattern similar to the mammalian MPR300. The LERP cytoplasmic domain shows highly conserved interactions with Drosophila and mammalian GGA adaptors known to mediate Golgi-endosome traffic of MPRs and other transmembrane cargo. Moreover, LERP rescues missorting of soluble lysosomal enzymes in MPR-deficient cells, giving strong evidence for a function that is equivalent to the mammalian counterpart. However, unlike the mammalian MPRs, LERP did not bind to the multimeric mannose 6-phosphate ligand phosphomannan. Thus ligand recognition by LERP does not depend on mannose 6-phosphate but may depend on a common feature present in mammalian lysosomal enzymes. Our data establish a potential important role for LERP in biogenesis of Drosophila lysosomes and suggest a GGA function also in the receptor-mediated lysosomal transport system in the fruit fly.