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.     

Neither type of mannose 6-phosphate receptor is sufficient for targeting of lysosomal enzymes along intracellular routes

Mouse embryonic fibroblasts that are deficient in the two mannose 6- phosphate receptors (MPRs) MPR 46 and MPR 300 missort the majority (> or = 85%) of soluble lysosomal proteins into the medium. Human MPR 46 and MPR 300 were expressed in these cells to test whether overexpression of a single type of MPR can restore transport of lysosomal proteins to lysosomes. Only a partial correction of the missorting was observed after overexpression of MPR 46. Even at MPR 46 levels that are five times higher than the wild-type level, more than one third of the newly synthesized lysosomal proteins accumulates in the secretions. Two-fold overexpression of MPR 300 completely corrects the missorting of lysosomal enzymes. However, at least one fourth of the lysosomal enzymes are transported along a secretion-recapture pathway that is sensitive to mannose 6-phosphate in medium. In control fibroblasts that express both types of MPR, the secretion-recapture pathway is of minor importance. These results imply that neither overexpression of MPR 46 nor MPR 300 is sufficient for targeting of lysosomal proteins along intracellular routes.     

Function and properties of chimeric MPR 46-MPR 300 mannose 6-phosphate receptors

The two known mannose 6-phosphate receptors (MPR 46 and MPR 300) mediate the transport of mannose 6-phosphate-containing lysosomal proteins to lysosomes. Endocytosis of extracellular mannose 6-phosphate ligands can only be mediated by MPR 300. Neither type of MPR appears to be sufficient for targetting the full complement of lysosomal enzymes to lysosomes. The complements of lysosomal enzymes transported by either of the two receptors are distinct but largely overlapping. Chimeric receptors were constructed in which the transmembrane and cytoplasmic domains of the two receptors were systematically exchanged. After expression of the chimeric receptors in cells lacking endogenous MPRs the binding of ligands, the subcellular distribution and the sorting efficiency for lysosomal enzymes were analyzed. All chimeras were functional, and their subcellular distribution was similar to that of wild type MPRs. The ability to endocytose lysosomal enzymes was restricted to receptors with the lumenal domain of MPR 300. The efficiency to sort lysosomal enzymes correlated with the lumenal and cytoplasmic domains of MPR 300. In contrast to the wild type receptors, a significant fraction of most of the chimeric receptors was misrouted to lysosomes, indicating that the signals determining the routing of MPRs have been fitted for the parent receptor polypeptides.     

Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor gene in mice results in misrouting of lysosomal proteins.

Lysosomal enzymes containing mannose 6-phosphate recognition markers are sorted to lysosomes by mannose 6-phosphate receptors (MPRs). The physiological importance of this targeting mechanism is illustrated by I-cell disease, a fatal lysosomal storage disorder caused by the absence of mannose 6-phosphate residues in lysosomal enzymes. Most mammalian cells express two MPRs. Although the binding specificities, subcellular distribution and expression pattern of the two receptors can be differentiated, their coexpression is not understood. The larger of the two receptors with an M(r) of approximately 300,000 (MPR300), which also binds IGFII, appears to have a dominant role in lysosomal enzyme targeting, while the function of the smaller receptor with an M(r) of 46,000 (MPR46) is less clear. To investigate the in vivo function of the MPR46, we generated MPR46-deficient mice using gene targeting in embryonic stem cells. Reduced intracellular retention of newly synthesized lysosomal proteins in cells from MPR46 -/- mice demonstrated an essential sorting function of MPR46. The phenotype of MPR46 -/- mice was normal, indicating mechanisms that compensate the MPR46 deficiency in vivo.     

The trafficking of prosaposin (SGP-1) and GM2AP to the lysosomes of TM4 Sertoli cells is mediated by sortilin and monomeric adaptor proteins

Prosaposin (SGP-1) and GM2 activator protein (GM2AP) are soluble sphingolipid activator proteins (SAPs) that are targeted to the lysosomal compartment of Sertoli cells to aid hydrolases in the breakdown of glycosphingolipids. To reach the lysosome, most soluble proteins must interact with the mannose 6-phosphate receptor (MPR). To be sorted from the Golgi, the MPR must bind to the Golgi associated, gamma-adaptin homologous, ARF binding proteins (GGAs), a group of monomeric adaptor proteins responsible for the recruitment of clathrin. It is well established, however, that the lysosomes of I-cell disease (ICD) patients have near normal levels of several lysosomal proteins, including prosaposin and GM2AP. ICD results from a mutation in the phosphotransferase that adds mannose 6-phosphate to hydrolases. Thus, prosaposin and GM2AP can traffic to lysosomes in a MPR independent manner. Previous work has demonstrated that an interaction with sphingomyelin in the Golgi membrane is necessary for the targeting of prosaposin by an unknown receptor. Using a TM4 Sertoli cell line, we tested the hypothesis that prosaposin and GM2AP are targeted to the lysosomal compartment via the sortilin receptor, which has been recently shown to have a GGA binding motif. Interestingly, dominant-negative GGAs, unable to bind clathrin to shuttle from the Golgi, prevented the trafficking of prosaposin and GM2AP to lysosomes. A dominant negative construct of sortilin lacking the GGA binding domain retained prosaposin and GM2AP in the Golgi. In conclusion, our results showed that the trafficking of prosaposin and GM2AP to the lysosome is dependent on sortilin.     

Drug-induced phospholipidosis is caused by blockade of mannose 6-phosphate receptor-mediated targeting of lysosomal enzymes

Cationic amphiphilic drugs (CADs) cause massive intracellular accumulation of phospholipids, thereby resulting in phospholipidosis (PLD); however, the molecular mechanism underlying CAD-induced PLD remains to be resolved. Here, we found that treatment of normal rat kidney cells with CADs known to induce PLD caused redistribution of a mannose 6-phosphate/IGF-II receptor (MPR300) from the TGN to endosomes and concomitantly increased the secretion of lysosomal enzymes, resulting in a decline of intracellular lysosomal enzyme levels. These results enable the interpretation of why CADs cause excessive accumulation of undegraded substrates, including phospholipids in lysosomes, and led to the conclusion that the impaired MPR300-mediated sorting system of lysosomal enzymes reflects the general mechanism of CAD-induced PLD. In addition, our findings suggest that the measurement of lysosomal enzyme activity secreted into culture medium is useful as a rapid and convenient in vitro early screening system to predict drugs that can induce PLD.     

Mannose 6-phosphate Receptor (MPR 300) Proteins from Goat and Chicken Bind Human IGF-II

Mannose 6-phosphate receptor proteins (MPR 300 and 46) in mammals have been shown to mediate transport of lysosomal enzymes to lysosomes intracellularly. Both receptors are also expressed on the plasma membrane. Only MPR 300 protein on the plasma membrane has been shown to be a multifunctional protein which in addition to binding mannose 6-phosphate containing proteins also binds human insulin-like growth factor-II (IGF-II) causing its internalization [Hille-Rehfeld, A. (1995) Mannose 6-phosphate receptors in sorting and transport of lysosomal enzymes. Biochim. Biophys. Acta. 1241: 177–194]. This property has been shown to be exhibited by other mammalian receptors but not by the chicken and frog receptors. In a recent study however it was shown that the fish embryo MPR 300 binds human IGF-II. [Mendez, E., Planas, J.V., Castillo, J., Navarro, I. and Gutierrez, J. (2001) Identification of a type II insulin-like growth factor receptor in fish embryos. Endocrinology, 142: 1090–1097]. In the present study, we demonstrate that the purified goat and chicken liver receptors bind human IGF-II by employing cross-linking experiments (purified receptors and radiolabeled IGF-II) and by ligand blotting (using purified receptors and biotinylated IGF-II). Further CEF cells (chicken embryonic fibroblasts) that are known to contain the putative MPR 300 protein were employed to demonstrate that the CEF cell receptor binds human IGF-II.     

Characterization of the mannose 6-phosphate receptor (Mr 300 kDa) protein dependent pathway of lysosomal enzyme targeting in Biomphalaria glabrata mollusc cells

Mammalian mannose 6-phosphate receptors (MPR 300 and 46) are involved in the targeting of newly synthesized lysosomal enzymes and only MPR 300 also participates in the endocytosis of various exogenous ligands. The present study describes for the first time the MPR 300 dependent pathway of lysosomal enzyme sorting in the Biomphalaria glabrata embryonic (Bge) cells. Lysosomal enzymes (arylsulfatase A, β-hexosaminidase and α-fucosidase) were identified by their enzymatic activities and by immunoprecipitation with specific antisera. Exposure of Bge cells to unio MPR 300 antiserum resulted in a dramatic loss of MPR 300 protein with a shortened half life of ∼20 min as compared to control cells exposed to preimmune serum in which the half life of MPR 300 was of ∼13 h. Loss of receptor proteins resulted in a significant misrouting of newly synthesized lysosomal enzymes and their secretion in cell culture medium as demonstrated by immunoprecipitation. The ability of Bge cells to uptake and internalize labeled arylsulfatase A, β-hexosaminidase and α-fucosidase enzymes contained in cell secretion products also indicated the role of B. glabrata MPR 300 (CIMPR) protein in internalization and targeting of lysosomal enzymes. M6P dependent binding of lysosomal enzymes to MPR 300 was shown by confocal microscopy and coimmunoprecipitation experiments.     

The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin

Most soluble lysosomal proteins bind the mannose 6-phosphate receptor (M6P-R) to be sorted to the lysosomes. However, the lysosomes of I-cell disease (ICD) patients, a condition resulting from a mutation in the phosphotransferase that adds mannose 6-phosphate to hydrolases, have near normal levels of several lysosomal proteins, including the sphingolipid activator proteins (SAPs), GM2AP and prosaposin. We tested the hypothesis that SAPs are targeted to the lysosomal compartment via the sortilin receptor. To test this hypothesis, a dominant-negative construct of sortilin and a sortilin small interfering RNA (siRNA) were introduced into COS-7 cells. Our results showed that both the truncated sortilin and the sortilin siRNA block the traffic of GM2AP and prosaposin to the lysosomal compartment. This observation was confirmed by a co-immunoprecipitation, which demonstrated that GM2AP and prosaposin are interactive partners of sortilin. Furthermore, a dominant-negative mutant GGA prevented the trafficking of prosaposin and GM2AP to lysosomes. In conclusion, our results show that the trafficking of SAPs is dependent on sortilin, demonstrating a novel lysosomal trafficking.     

Lysosomal integral membrane glycoproteins are expressed at high levels in the inclusion bodies of I-cell disease fibroblasts

The localization, expression, and transport of two lysosomal integral membrane glycoproteins of human cells, hLAMP-1 and hLAMP-2, have been studied in mucolipidosis II (I-cell disease) fibroblasts. These cells are deficient in N-acetylglucosaminylphosphotransferase, one of the enzymes required for addition of the mannose 6-phosphate recognition signal to newly synthesized lysosomal hydrolases and a prerequisite for the sorting and transport of the hydrolases to lysosomes. I-cells analyzed by immunofluorescence microscopy with monoclonal antibodies against hLAMP-1 and hLAMP-2 showed intense staining of the inclusion bodies covering most of the cytoplasm of the cells. Immunoelectron microscopy confirmed this localization and showed that the hLAMP-positive vesicles commonly contained membrane structures or electron-dense homogeneous material characteristic of secondary lysosomes. Studies of the biosynthesis of hLAMP-2 in I-cells pulse-labeled with [35S]methionine indicated that the molecule is glycosylated in the Golgi system, is transported to vesicles with the high density characteristic of lysosomes, and has chemical properties similar to those of the glycoprotein synthesized in normal cells. The concentration of the hLAMP-2 glycoprotein was three- to fourfold greater than that in normal fibroblasts, in sharp contrast to the reduced levels of lysosomal hydrolases seen in I-cells. These experiments demonstrate that the inclusion bodies in I-cells have properties of secondary lysosomes and that the transport and targeting of the lysosomal membrane glycoproteins to the inclusion bodies of these cells is not coupled to the mannose 6-phosphate system for transporting soluble acid hydrolases.     

Identification of novel lysosomal matrix proteins by proteome analysis

The lysosomal matrix is estimated to contain about 50 different proteins. Most of the matrix proteins are acid hydrolases that depend on mannose 6-phosphate receptors (MPR) for targeting to lysosomes. Here, we describe a comprehensive proteome analysis of MPR-binding proteins from mouse. Mouse embryonic fibroblasts defective in both MPR (MPR 46-/- and MPR 300-/-) are known to secrete the lysosomal matrix proteins. Secretions of these cells were affinity purified using an affinity matrix derivatized with MPR46 and MPR300. In the protein fraction bound to the affinity matrix and eluted with mannose 6-phosphate, 34 known lysosomal matrix proteins, 4 candidate proteins of the lysosomal matrix and 4 non-lysosomal contaminants were identified by mass spectrometry after separation by two-dimensional gel electrophoresis or by multidimensional protein identification technology. For 3 of the candidate proteins, mammalian ependymin-related protein-2 (MERP-2), retinoid-inducible serine carboxypeptidase (RISC) and the hypothetical 66.3-kDa protein we could verify that C-terminally tagged forms bound in an M6P-dependent manner to an MPR-affinity matrix and were internalized via MPR-mediated endocytosis. Hence these 3 proteins are likely to represent hitherto unrecognized lysosomal matrix proteins.     

Mannose 6-phosphate receptors, Niemann-Pick C2 protein, and lysosomal cholesterol accumulation

Niemann-Pick disease type C (NPC), caused by mutations in the NPC1 gene or the NPC2 gene, is characterized by the accumulation of unesterified cholesterol and other lipids in endo/lysosomal compartments. NPC2 is a small, soluble, lysosomal protein that is targeted to this compartment via a mannose 6-phosphate-inhibitable pathway. To obtain insight into the roles of mannose 6-phosphate receptors (MPRs) in NPC2 targeting, we here examine the trafficking and function of NPC2 in fibroblast lines deficient in one or both of the two MPRs, MPR46 and MPR300. We demonstrate that either MPR alone is sufficient to transport NPC2 to the endo/lysosomal compartment, although MPR300 seems to be more efficient than MPR46. In the absence of both MPRs, NPC2 is secreted into the culture medium, and only a small amount of intracellular NPC2 can be detected, mainly in the endoplasmic reticulum. This leads to massive accumulation of unesterified cholesterol in the endo/lysosomal compartment of the MPR46/300-deficient fibroblasts, a phenotype similar to that of the NPC patient fibroblasts. In addition, we observed an upregulation of NPC1 protein and mRNA in the MPR-double-deficient cells. Taken together, our results suggest that the lysosomal targeting of NPC2 is strictly dependent on MPRs in fibroblasts.     

The yeast Vps10p cytoplasmic tail mediates lysosomal sorting in mammalian cells and interacts with human GGAs.

Yeast Vps10p is a receptor for transport of the soluble vacuolar hydrolase carboxypeptidase Y to the lysosome-like vacuole. Its functional equivalents in mammalian cells are the mannose 6-phosphate receptors that mediate sorting to lysosomes of mannose 6-phosphate-containing lysosomal proteins. A chimeric receptor was constructed by substituting the cytoplasmic domain of M(r) 300,000 mannose 6-phosphate receptor with the Vps10p cytoplasmic tail. Expression of the chimera in cells lacking endogenous mannose 6-phosphate receptors resulted in a subcellular receptor distribution and an efficiency in sorting of lysosomal enzymes similar to that of the wild type M(r) 300,000 mannose 6-phosphate receptor. Moreover, the cytoplasmic tail of the Vps10p was found to interact with GGA1 and GGA2, two mammalian members of a recently discovered family of clathrin-binding cytosolic proteins that participate in trans-Golgi network-endosome trafficking in both mammals and yeast. Our findings suggest a conserved machinery for Golgi-endosome/vacuole sorting and may serve as a model for future studies of yeast proteins.     

P-type lectins

The two members of the P-type lectin family, the cation-dependent mannose 6-phosphate receptor (CD-MPR) and the insulin-like growth factor II/mannose 6-phosphate receptor (IGF-II/MPR), are distinguished from all other lectins by their ability to recognize phosphorylated mannose residues. The P-type lectins play an essential role in the generation of functional lysosomes within the cells of higher eukaryotes by directing newly synthesized lysosomal enzymes bearing the mannose 6-phosphate (M6P) signal to lysosomes. At the cell surface, the IGF-II/MPR also binds to the nonglycosylated polypeptide hormone, IGF-II, targeting this potent mitogenic factor for degradation in lysosomes. Moreover, in recent years, the multifunctional nature of the IGF-II/MPR has become increasingly apparent, as the list of extracellular ligands recognized by this receptor has grown to include a diverse spectrum of M6P-containing proteins as well as nonglycosylated ligands, implicating a role for the IGF-II/MPR in a number of important physiological pathways. Recent investigations have provided valuable insights into the molecular basis of ligand recognition by the MPRs as well as the complex intracellular trafficking pathways traversed by these receptors. This review provides a current view on the structures, functions, and medical relevance of the P-type lectins.     

Lysosomal storage causes cellular dysfunction in mucolipidosis II skin fibroblasts

Mucolipidosis II (ML-II) is a fatal inherited metabolic disease caused by deficiency of GlcNAc-phosphotransferase, which plays a role in generating the mannose 6-phosphate recognition marker on lysosomal enzymes. In ML-II, many lysosomal acid hydrolases are mistargeted out of cells, and lysosomes become filled with undigested substrates, which explains inclusion cell disease as an alternative name for this disease. In this study, we revealed various cellular phenotypes in ML-II skin fibroblasts. We quantitated phospholipid and cholesterol within cells and showed ~2-fold accumulation in ML-II as compared with normal cells. Lysosomal pH of ML-II cells was higher than that of normal cells (5.29 ± 0.08 versus 4.79 ± 0.10, p < 0.001). The proliferated lysosomes in ML-II cells were accumulated ~3-fold in amount as compared with normal cells. Intracellular logistics including endocytosis and mannose 6-phosphate receptor recycling were impaired in ML-II cells. To confirm whether these ML-II cellular phenotypes derive from deficient lysosomal acid hydrolases within lysosomes, we performed supplementation of lysosomal enzymes using a partially purified total enzyme mixture, which was derived from the conditioned culture medium of normal skin fibroblasts after NH(4)Cl treatment. This supplementation corrected all of the previously described ML-II phenotypes. In addition, the autophagic and mitochondrial impairment that we have previously reported improved, and inclusion bodies disappeared on electron micrography following total lysosomal enzyme supplementation. Our results indicate that various cellular phenotypes in ML-II are caused by the deficiency of many lysosomal enzymes and massive accumulation of undigested substrates.     

Distribution and trafficking of MPR300 is normal in cells with cholesterol accumulated in late endocytic compartments: evidence for early endosome-to-TGN trafficking of MPR300

It has been reported that an accumulation of cholesterol within late endosomes/lysosomes in Niemann-Pick type C (NPC) fibroblasts and U18666A-treated cells causes impairment of retrograde trafficking of the cation-independent mannose 6-phosphate/IGF-II receptor (MPR300) from late endosomes to the trans-Golgi network (TGN). In apparent conflict with these results, here we show that as in normal fibroblasts, MPR300 localizes exclusively to the TGN in NPC fibroblasts as well as in normal fibroblasts treated with U18666A. This localization can explain why several lysosomal properties and functions, such as intracellular lysosomal enzyme activity and localization, the biosynthesis of cathepsin D, and protein degradation, are all normal in NPC fibroblasts. These results, therefore, suggest that the accumulation of cholesterol in late endosomes/lysosomes does not affect the retrieval of MPR300 from endosomes to the TGN. Furthermore, treatment of normal and NPC fibroblasts with chloroquine, which inhibits membrane traffic from early endosomes to the TGN, resulted in a redistribution of MPR300 to EEA1 and internalized transferrin-positive, but LAMP-2-negative, early-recycling endosomes. We propose that in normal and NPC fibroblasts, MPR300 is exclusively targeted from the TGN to early endosomes, from where it rapidly recycles back to the TGN without being delivered to late endosomes. This notion provides important insights into the definition of late endosomes, as well as the biogenesis of lysosomes.     

Mannose-6-phosphate receptors (MPR 300 and 46) from the highly evolved invertebrate <Emphasis Type="Italic">Asterias rubens</Emphasis> (Echinodermate): biochemical and functional characterization of MPR 46 protein

Mammalian mannose 6-phosphate receptors (MPR 300 and 46) mediate transport of lysosomal enzymes to lysosomes. Recent studies established that the receptors are conserved throughout vertebrates. Although we purified the mollusc receptors and identified only a lysosomal enzyme receptor protein (LERP) in the Drosophila melanogaster, little is known about their structure and functional roles in the invertebrates. In the present study, we purified the putative receptors from the highly evolved invertebrate, starfish, cloned the cDNA for the MPR 46, and expressed it in mpr^(−/−) mouse embryonic fibroblast cells. Structural comparison of starfish receptor sequences with other vertebrate receptors gave valuable information on its extensive structural homology with the vertebrate MPR 46 proteins. The expressed protein efficiently sorts lysosomal enzymes within the cells establishing a functional role for this protein. This first report on the invertebrate MPR 46 further confirms the structural and functional conservation of the receptor not only in the vertebrates but also in the invertebrates.     

Mr 46,000 mannose 6-phosphate specific receptor: its role in targeting of lysosomal enzymes.

Antibodies that block the ligand binding site of the cation-dependent mannose 6-phosphate specific receptor (Mr 46,000 MPR) were used to probe the function of the receptor in transport of lysosomal enzymes. Addition of the antibodies to the medium of Morris hepatoma 7777 cells, which express only the Mr 46,000 MPR, resulted in a decreased intracellular retention and increased secretion of newly synthesized lysosomal enzymes. In fibroblasts and HepG2 cells that express the cation-independent mannose 6-phosphate specific receptor (Mr 215,000 MPR) in addition to the Mr 46,000 MPR, antibodies against the Mr 46,000 MPR inhibited the intracellular retention of newly synthesized lysosomal enzymes only when added to the medium together with antibodies against the Mr 215,000 MPR. Morris hepatoma (M.H.) 7777 did not endocytose lysosomal enzymes, while U937 monocytes, which express both types of MPR, internalized lysosomal enzymes. The uptake was inhibited by antibodies against the Mr 215,000 MPR, but not by antibodies against the Mr 46,000 MPR. These observations suggest that Mr 46,000 MPR mediates transport of endogenous but not endocytosis of exogenous lysosomal enzymes. Internalization of receptor antibodies indicated that the failure to mediate endocytosis of lysosomal enzymes is due to an inability of surface Mr 46,000 MPR to bind ligands rather than its exclusion from the plasma membrane or from internalization.     

Purification and biochemical characterization of a lysosomal α-fucosidase from the deuterostomia Asterias rubens

In vertebrates, mannose 6-phosphate receptors [MPR300 (Mr 300 kDa) and MPR46 (Mr 46 kDa)] are highly conserved transmembrane glycoproteins that mediate transport of lysosomal enzymes to lysosomes. Our studies have revealed the appearance of these putative receptors in invertebrates such as the molluscs and deuterostomes. Starfish tissue extracts contain several lysosomal enzyme activities and here we describe the affinity purification of α-fucosidase. The purified enzyme is a glycoprotein that exhibited a molecular mass of ∼56 kDa in SDS-PAGE under reducing conditions. It has also cross-reacted with an antiserum to the mollusc enzyme suggesting antigenic similarities among the two invertebrate enzymes. LC–MS/MS analysis of the proteolytic peptides of the purified enzyme in combination with de novo sequencing allowed us to do partial amino acid sequence determination of the enzyme. These data suggest that this invertebrate enzyme is homologous to the known mammalian enzyme. The purified enzyme exhibited a mannose 6-phosphate dependent interaction with the immobilized starfish MPR300 protein. Our results demonstrate that the lysosomal enzyme targeting pathway is conserved even among the invertebrates.