AAV-mediated gene therapy for sialidosis.

Sialidosis (mucolipidosis I) is a glycoprotein storage disease, clinically characterized by a spectrum of systemic and neurological phenotypes. The primary cause of the disease is deficiency of the lysosomal sialidase NEU1, resulting in accumulation of sialylated glycoproteins/oligosaccharides in tissues and body fluids. Neu1-/- mice recapitulate the severe, early-onset forms of the disease, affecting visceral organs, muscles, and the nervous system, with widespread lysosomal vacuolization evident in most cell types. Sialidosis is considered an orphan disorder with no therapy currently available. Here, we assessed the therapeutic potential of AAV-mediated gene therapy for the treatment of sialidosis. Neu1-/- mice were co-injected with two scAAV2/8 vectors, expressing human NEU1 and its chaperone PPCA. Treated mice were phenotypically indistinguishable from their WT controls. NEU1 activity was restored to different extent in most tissues, including the brain, heart, muscle, and visceral organs. This resulted in diminished/absent lysosomal vacuolization in multiple cell types and reversal of sialyl-oligosacchariduria. Lastly, normalization of lysosomal exocytosis in the cerebrospinal fluids and serum of treated mice, coupled to diminished neuroinflammation, were measures of therapeutic efficacy. These findings point to AAV-mediated gene therapy as a suitable treatment for sialidosis and possibly other diseases, associated with low NEU1 expression.

Haploinsufficiency of the lysosomal sialidase NEU1 results in a model of pleomorphic rhabdomyosarcoma in mice.

Rhabdomyosarcoma, the most common pediatric sarcoma, has no effective treatment for the pleomorphic subtype. Still, what triggers transformation into this aggressive phenotype remains poorly understood. Here we used Ptch1+/-/ETV7TG/+/- mice with enhanced incidence of rhabdomyosarcoma to generate a model of pleomorphic rhabdomyosarcoma driven by haploinsufficiency of the lysosomal sialidase neuraminidase 1. These tumors share mostly features of embryonal and some of alveolar rhabdomyosarcoma. Mechanistically, we show that the transforming pathway is increased lysosomal exocytosis downstream of reduced neuraminidase 1, exemplified by the redistribution of the lysosomal associated membrane protein 1 at the plasma membrane of tumor and stromal cells. Here we exploit this unique feature for single cell analysis and define heterogeneous populations of exocytic, only partially differentiated cells that force tumors to pleomorphism and promote a fibrotic microenvironment. These data together with the identification of an adipogenic signature shared by human rhabdomyosarcoma, and likely fueling the tumor's metabolism, make this model of pleomorphic rhabdomyosarcoma ideal for diagnostic and therapeutic studies.

Distinct functions of dimeric and monomeric scaffold protein Alix in regulating F-actin assembly and loading of exosomal cargo.

Alix is a ubiquitously expressed scaffold protein that participates in numerous cellular processes related to the remodeling/repair of membranes and the actin cytoskeleton. Alix exists in monomeric and dimeric/multimeric configurations, but how dimer formation occurs and what role the dimer has in Alix-mediated processes are still largely elusive. Here, we reveal a mechanism for Alix homodimerization mediated by disulfide bonds under physiological conditions and demonstrate that the Alix dimer is enriched in exosomes and F-actin cytoskeleton subcellular fractions. Proteomic analysis of exosomes derived from Alix-/- primary cells underlined the indispensable role of Alix in loading syntenin into exosomes, thereby regulating the cellular levels of this protein. Using a set of deletion mutants, we define the function of Alix Bro1 domain, which is solely required for its exosomal localization, and that of the V domain, which is needed for recruiting syntenin into exosomes. We reveal an essential role for Cys814 within the disordered proline-rich domain for Alix dimerization. By mutating this residue, we show that Alix remains exclusively monomeric and, in this configuration, is effective in loading syntenin into exosomes. In contrast, loss of dimerization affects the ability of Alix to associate with F-actin, thereby compromising Alix-mediated cytoskeleton remodeling. We propose that dimeric and monomeric forms of Alix selectively execute two of the protein's main functions: exosomal cargo loading and cytoskeleton remodeling.

Lysosomes and Cancer Progression: A Malignant Liaison.

During primary tumorigenesis isolated cancer cells may undergo genetic or epigenetic changes that render them responsive to additional intrinsic or extrinsic cues, so that they enter a transitional state and eventually acquire an aggressive, metastatic phenotype. Among these changes is the alteration of the cell metabolic/catabolic machinery that creates the most permissive conditions for invasion, dissemination, and survival. The lysosomal system has emerged as a crucial player in this malignant transformation, making this system a potential therapeutic target in cancer. By virtue of their ubiquitous distribution in mammalian cells, their multifaced activities that control catabolic and anabolic processes, and their interplay with other organelles and the plasma membrane (PM), lysosomes function as platforms for inter- and intracellular communication. This is due to their capacity to adapt and sense nutrient availability, to spatially segregate specific functions depending on their position, to fuse with other compartments and with the PM, and to engage in membrane contact sites (MCS) with other organelles. Here we review the latest advances in our understanding of the role of the lysosomal system in cancer progression. We focus on how changes in lysosomal nutrient sensing, as well as lysosomal positioning, exocytosis, and fusion perturb the communication between tumor cells themselves and between tumor cells and their microenvironment. Finally, we describe the potential impact of MCS between lysosomes and other organelles in propelling cancer growth and spread.

Transcription factor competition regulates lysosomal biogenesis and autophagy.

"In the field of observation, chance favours only the prepared mind" (Louis Pasteur). This motto seems to have guided our unexpected results published recently in Nature Communications, where we describe an epigenetic rheostat that regulates expression of the constituents of the lysosomal and autophagic systems.

MYC competes with MiT/TFE in regulating lysosomal biogenesis and autophagy through an epigenetic rheostat.

Coordinated regulation of the lysosomal and autophagic systems ensures basal catabolism and normal cell physiology, and failure of either system causes disease. Here we describe an epigenetic rheostat orchestrated by c-MYC and histone deacetylases that inhibits lysosomal and autophagic biogenesis by concomitantly repressing the expression of the transcription factors MiT/TFE and FOXH1, and that of lysosomal and autophagy genes. Inhibition of histone deacetylases abates c-MYC binding to the promoters of lysosomal and autophagy genes, granting promoter occupancy to the MiT/TFE members, TFEB and TFE3, and/or the autophagy regulator FOXH1. In pluripotent stem cells and cancer, suppression of lysosomal and autophagic function is directly downstream of c-MYC overexpression and may represent a hallmark of malignant transformation. We propose that, by determining the fate of these catabolic systems, this hierarchical switch regulates the adaptive response of cells to pathological and physiological cues that could be exploited therapeutically.

Excessive exosome release is the pathogenic pathway linking a lysosomal deficiency to generalized fibrosis.

Lysosomal exocytosis is a ubiquitous process negatively regulated by neuraminidase 1 (NEU1), a sialidase mutated in the glycoprotein storage disease sialidosis. In Neu1-/- mice, excessive lysosomal exocytosis is at the basis of disease pathogenesis. Yet, the tissue-specific molecular consequences of this deregulated pathway are still unfolding. We now report that in muscle connective tissue, Neu1-/- fibroblasts have features of myofibroblasts and are proliferative, migratory, and exocytose large amounts of exosomes. These nanocarriers loaded with activated transforming growth factor-ß and wingless-related integration site (WNT)/ß-catenin signaling molecules propagate fibrotic signals to other cells, maintaining the tissue in a prolonged transitional status. Myofibroblast-derived exosomes fed to normal fibroblasts convert them into myofibroblasts, changing the recipient cells' proliferative and migratory properties. These findings reveal an unexpected exosome-mediated signaling pathway downstream of NEU1 deficiency that propagates a fibrotic disease and could be implicated in idiopathic forms of fibrosis in humans.

Lysosomal storage diseases.

Lysosomal storage diseases (LSDs) are a group of over 70 diseases that are characterized by lysosomal dysfunction, most of which are inherited as autosomal recessive traits. These disorders are individually rare but collectively affect 1 in 5,000 live births. LSDs typically present in infancy and childhood, although adult-onset forms also occur. Most LSDs have a progressive neurodegenerative clinical course, although symptoms in other organ systems are frequent. LSD-associated genes encode different lysosomal proteins, including lysosomal enzymes and lysosomal membrane proteins. The lysosome is the key cellular hub for macromolecule catabolism, recycling and signalling, and defects that impair any of these functions cause the accumulation of undigested or partially digested macromolecules in lysosomes (that is, 'storage') or impair the transport of molecules, which can result in cellular damage. Consequently, the cellular pathogenesis of these diseases is complex and is currently incompletely understood. Several LSDs can be treated with approved, disease-specific therapies that are mostly based on enzyme replacement. However, small-molecule therapies, including substrate reduction and chaperone therapies, have also been developed and are approved for some LSDs, whereas gene therapy and genome editing are at advanced preclinical stages and, for a few disorders, have already progressed to the clinic.

Palmitoylation is a post-translational modification of Alix regulating the membrane organization of exosome-like small extracellular vesicles.

BACKGROUND: Virtually all cell types have the capacity to secrete nanometer-sized extracellular vesicles, which have emerged in recent years as potent signal transducers and cell-cell communicators. The multifunctional protein Alix is a bona fide exosomal regulator and skeletal muscle cells can release Alix-positive nano-sized extracellular vesicles, offering a new paradigm for understanding how myofibers communicate within skeletal muscle and with other organs. S-palmitoylation is a reversible lipid post-translational modification, involved in different biological processes, such as the trafficking of membrane proteins, achievement of stable protein conformations, and stabilization of protein interactions. METHODS: Here, we have used an integrated biochemical-biophysical approach to determine whether S-palmitoylation contributes to the regulation of extracellular vesicle production in skeletal muscle cells. RESULTS: We ascertained that Alix is S-palmitoylated and that this post-translational modification influences its protein-protein interaction with CD9, a member of the tetraspanin protein family. Furthermore, we showed that the structural organization of the lipid bilayer of the small (nano-sized) extracellular vesicle membrane with altered palmitoylation is qualitatively different compared to mock control vesicles. CONCLUSIONS: We propose that S-palmitoylation regulates the function of Alix in facilitating the interactions among extracellular vesicle-specific regulators and maintains the proper structural organization of exosome-like extracellular vesicle membranes. GENERAL SIGNIFICANCE: Beyond its biological relevance, our study also provides the means for a comprehensive structural characterization of EVs.

Alix-mediated assembly of the actomyosin-tight junction polarity complex preserves epithelial polarity and epithelial barrier.

Maintenance of epithelial cell polarity and epithelial barrier relies on the spatial organization of the actin cytoskeleton and proper positioning/assembly of intercellular junctions. However, how these processes are regulated is poorly understood. Here we reveal a key role for the multifunctional protein Alix in both processes. In a knockout mouse model of Alix, we identified overt structural changes in the epithelium of the choroid plexus and in the ependyma, such as asymmetrical cell shape and size, misplacement and abnormal beating of cilia, blebbing of the microvilli. These defects culminate in excessive cell extrusion, enlargement of the lateral ventricles and hydrocephalus. Mechanistically, we find that by interacting with F-actin, the Par complex and ZO-1, Alix ensures the formation and maintenance of the apically restricted actomyosin-tight junction complex. We propose that in this capacity Alix plays a role in the establishment of apical-basal polarity and in the maintenance of the epithelial barrier.

Loss of Cellular Sialidases Does Not Affect the Sialylation Status of the Prion Protein but Increases the Amounts of Its Proteolytic Fragment C1.

The central molecular event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrPC), which is a sialoglycoprotein, into the disease-associated, transmissible form denoted PrPSc. Recent studies revealed a correlation between the sialylation status of PrPSc and incubation time to disease and introduced a new hypothesis that progression of prion diseases could be controlled or reversed by altering the sialylation level of PrPC. Of the four known mammalian sialidases, the enzymes that cleave off sialic acid residues, only NEU1, NEU3 and NEU4 are expressed in the brain. To test whether cellular sialidases control the steady-state sialylation level of PrPC and to identify the putative sialidase responsible for desialylating PrPC, we analyzed brain-derived PrPC from knockout mice deficient in Neu1, Neu3, Neu4, or from Neu3/Neu4 double knockouts. Surprisingly, no differences in the sialylation of PrPC or its proteolytic product C1 were noticed in any of the knockout mice tested as compared to the age-matched controls. However, significantly higher amounts of the C1 fragment relative to full-length PrPC were detected in the brains of Neu1 knockout mice as compared to WT mice or to the other knockout mice. Additional experiments revealed that in neuroblastoma cell line the sialylation pattern of C1 could be changed by an inhibitor of sialylatransferases. In summary, this study suggests that targeting cellular sialidases is apparently not the correct strategy for altering the sialylation levels of PrPC, whereas modulating the activity of sialylatransferases might offer a more promising approach. Our findings also suggest that catabolism of PrPC involves its α-cleavage followed by desialylation of the resulting C1 fragments by NEU1 and consequent fast degradation of the desialylated products.

Bis(monoacylglycero)phosphate: a secondary storage lipid in the gangliosidoses.

Bis(monoacylglycero)phosphate (BMP) is a negatively charged glycerophospholipid with an unusual sn-1;sn-1' structural configuration. BMP is primarily enriched in endosomal/lysosomal membranes. BMP is thought to play a role in glycosphingolipid degradation and cholesterol transport. Elevated BMP levels have been found in many lysosomal storage diseases (LSDs), suggesting an association with lysosomal storage material. The gangliosidoses are a group of neurodegenerative LSDs involving the accumulation of either GM1 or GM2 gangliosides resulting from inherited deficiencies in ß-galactosidase or ß-hexosaminidase, respectively. Little information is available on BMP levels in gangliosidosis brain tissue. Our results showed that the content of BMP in brain was significantly greater in humans and in animals (mice, cats, American black bears) with either GM1 or GM2 ganglioside storage diseases, than in brains of normal subjects. The storage of BMP and ganglioside GM2 in brain were reduced similarly following adeno-associated viral-mediated gene therapy in Sandhoff disease mice. We also found that C22:6, C18:0, and C18:1 were the predominant BMP fatty acid species in gangliosidosis brains. The results show that BMP accumulates as a secondary storage material in the brain of a broad range of mammals with gangliosidoses.

Regulated lysosomal exocytosis mediates cancer progression.

Understanding how tumor cells transition to an invasive and drug-resistant phenotype is central to cancer biology, but the mechanisms underlying this transition remain unclear. We show that sarcomas gain these malignant traits by inducing lysosomal exocytosis, a ubiquitous physiological process. During lysosomal exocytosis, the movement of exocytic lysosomes along the cytoskeleton and their docking at the plasma membrane involve LAMP1, a sialylated membrane glycoprotein and target of the sialidase NEU1. Cleavage of LAMP1 sialic acids by NEU1 limits the extent of lysosomal exocytosis. We found that by down-regulation of NEU1 and accumulation of oversialylated LAMP1, tumor cells exacerbate lysosomal exocytosis of soluble hydrolases and exosomes. This facilitates matrix invasion and propagation of invasive signals, and purging of lysosomotropic chemotherapeutics. In Arf (-/-) mice, Neu1 haploinsufficiency fostered the development of invasive, pleomorphic sarcomas, expressing epithelial and mesenchymal markers, and lysosomal exocytosis effectors, LAMP1 and Myosin-11. These features are analogous to those of metastatic, pleomorphic human sarcomas, where low NEU1 levels correlate with high expression of lysosomal exocytosis markers. In a therapeutic proof of principle, we demonstrate that inhibiting lysosomal exocytosis reversed invasiveness and chemoresistance in aggressive sarcoma cells. Thus, we reveal that this unconventional, lysosome-regulated pathway plays a primary role in tumor progression and chemoresistance.

Broad and direct interaction between TLR and Siglec families of pattern recognition receptors and its regulation by Neu1.

Both pathogen- and tissue damage-associated molecular patterns induce inflammation through toll-like receptors (TLRs), while sialic acid-binding immunoglobulin superfamily lectin receptors (Siglecs) provide negative regulation. Here we report extensive and direct interactions between these pattern recognition receptors. The promiscuous TLR binders were human SIGLEC-5/9 and mouse Siglec-3/E/F. Mouse Siglec-G did not show appreciable binding to any TLRs tested. Correspondingly, Siglece deletion enhanced dendritic cell responses to all microbial TLR ligands tested, while Siglecg deletion did not affect the responses to these ligands. TLR4 activation triggers Neu1 translocation to cell surface to disrupt TLR4:Siglec-E interaction. Conversely, sialidase inhibitor Neu5Gc2en prevented TLR4 ligand-induced disruption of TLR4:Siglec E/F interactions. Absence of Neu1 in hematopoietic cells or systematic treatment with sialidase inhibitor Neu5Gc2en protected mice against endotoxemia. Our data raised an intriguing possibility of a broad repression of TLR function by Siglecs and a sialidase-mediated de-repression that allows positive feedback of TLR activation during infection.

Lysosomal NEU1 deficiency affects amyloid precursor protein levels and amyloid-ß secretion via deregulated lysosomal exocytosis.

Alzheimer's disease (AD) belongs to a category of adult neurodegenerative conditions, which are associated with intracellular and extracellular accumulation of neurotoxic protein aggregates. Understanding how these aggregates are formed, secreted and propagated by neurons has been the subject of intensive research, but so far no preventive or curative therapy for AD is available, and clinical trials have been largely unsuccessful. Here we show that deficiency of the lysosomal sialidase NEU1 leads to the spontaneous occurrence of an AD-like amyloidogenic process in mice. This involves two consecutive events linked to NEU1 loss-of-function--accumulation and amyloidogenic processing of an oversialylated amyloid precursor protein in lysosomes, and extracellular release of Aß peptides by excessive lysosomal exocytosis. Furthermore, cerebral injection of NEU1 in an established AD mouse model substantially reduces ß-amyloid plaques. Our findings identify an additional pathway for the secretion of Aß and define NEU1 as a potential therapeutic molecule for AD.

Chaperone-mediated gene therapy with recombinant AAV-PPCA in a new mouse model of type I sialidosis.

The lysosomal storage disease sialidosis is caused by a primary deficiency of the sialidase N-acetyl-α-neuraminidase-1 (NEU1). Patients with type I sialidosis develop an attenuated, non-neuropathic form of the disease also named cherry red spot myoclonus syndrome, with symptoms arising during juvenile/ adult age. NEU1 requires binding to its chaperone, protective protein/cathepsin A (PPCA), for lysosomal compartmentalization, stability and catalytic activation. We have generated a new mouse model of type I sialidosis that ubiquitously expresses a NEU1 variant carrying a V54M amino acid substitution identified in an adult patient with type I sialidosis. Mutant mice developed signs of lysosomal disease after 1year of age, predominantly in the kidney, albeit low residual NEU1 activity was detected in most organs and cell types. We demonstrate that the activity of the mutant enzyme could be effectively increased in all systemic tissues by chaperone-mediated gene therapy with a liver-tropic recombinant AAV2/8 vector expressing PPCA. This resulted in clear amelioration of the disease phenotype. These results suggest that at least some of the NEU1 mutations associated with type I sialidosis may respond to PPCA-chaperone-mediated gene therapy.

Emphysema in an adult with galactosialidosis linked to a defect in primary elastic fiber assembly.

Galactosialidosis is a lysosomal storage disorder caused by loss of function of protective protein cathepsin A, which leads to secondary deficiencies of ß-galactosidase and neuraminidase-1. Emphysema has not been previously reported as a possible complication of this disorder, but we now describe this condition in a 41-year-old, non-smoking male. Our patient did not display deficiency in α-1-antitrypsin, the most common cause of emphysema in non-smokers, which brings about disseminated elastolysis. We therefore hypothesized that loss of cathepsin A activity was responsible because of previously published evidence showing it is prerequisite for normal elastogenesis. We now present experimental evidence to support this theory by demonstrating impaired primary elastogenesis in cultures of dermal fibroblasts from our patient. The obtained data further endorse our previous finding that functional integrity of the cell surface-targeted molecular complex of cathepsin A, neuraminidase-1 and the elastin-binding protein (spliced variant of ß-galactosidase) is prerequisite for the normal assembly of elastic fibers. Importantly, we also found that elastic fiber production was increased after exposure either to losartan, spironolactone, or dexamethasone. Of immediate clinical relevance, our data suggest that surviving patients with galactosialidosis should have periodic assessment of their pulmonary function. We also encourage further experimental exploration of therapeutic potential of the afore-mentioned elastogenesis-stimulating drugs for the alleviation of pathological processes in galactosialidosis that could be mechanistically linked to impaired deposition of elastic fibers.

Preclinical dose-finding study with a liver-tropic, recombinant AAV-2/8 vector in the mouse model of galactosialidosis.

Galactosialidosis (GS) is a lysosomal storage disease linked to deficiency of the protective protein/cathepsin A (PPCA). Similarly to GS patients, Ppca-null mice develop a systemic disease of the reticuloendothelial system, affecting most visceral organs and the nervous system. Symptoms include severe nephropathy, visceromegaly, infertility, progressive ataxia, and shortened life span. Here, we have conducted a preclinical, dose-finding study on a large cohort of GS mice injected intravenously at 1 month of age with increasing doses of a GMP-grade rAAV2/8 vector, expressing PPCA under the control of a liver-specific promoter. Treated mice, monitored for 16 weeks post-treatment, had normal physical appearance and behavior without discernable side effects. Despite the restricted expression of the transgene in the liver, immunohistochemical and biochemical analyses of other systemic organs, serum, and urine showed a dose-dependent, widespread correction of the disease phenotype, suggestive of a protein-mediated mechanism of cross-correction. A notable finding was that rAAV-treated GS mice showed high expression of PPCA in the reproductive organs, which resulted in reversal of their infertility. Together these results support the use of this rAAV-PPCA vector as a viable and safe method of gene delivery for the treatment of systemic disease in non-neuropathic GS patients.

AAV-mediated gene delivery in adult GM1-gangliosidosis mice corrects lysosomal storage in CNS and improves survival.

BACKGROUND: GM1-gangliosidosis is a glycosphingolipid (GSL) lysosomal storage disease caused by a genetic deficiency of acid ß-galactosidase (ßgal), which results in the accumulation of GM1-ganglioside and its asialo-form (GA1) primarily in the CNS. Age of onset ranges from infancy to adulthood, and excessive ganglioside accumulation produces progressive neurodegeneration and psychomotor retardation in humans. Currently, there are no effective therapies for the treatment of GM1-gangliosidosis. METHODOLOGY/PRINCIPAL FINDINGS: In this study we examined the effect of thalamic infusion of AAV2/1-ßgal vector in adult GM1 mice on enzyme distribution, activity, and GSL content in the CNS, motor behavior, and survival. Six to eight week-old GM1 mice received bilateral injections of AAV vector in the thalamus, or thalamus and deep cerebellar nuclei (DCN) with pre-determined endpoints at 1 and 4 months post-injection, and the humane endpoint, or 52 weeks of age. Enzyme activity was elevated throughout the CNS of AAV-treated GM1 mice and GSL storage nearly normalized in most structures analyzed, except in the spinal cord which showed ∼50% reduction compared to age-matched untreated GM1 mice spinal cord. Survival was significantly longer in AAV-treated GM1 mice (52 wks) than in untreated mice. However the motor performance of AAV-treated GM1 mice declined over time at a rate similar to that observed in untreated GM1 mice. CONCLUSIONS/SIGNIFICANCE: Our studies show that the AAV-modified thalamus can be used as a 'built-in' central node network for widespread distribution of lysosomal enzymes in the mouse cerebrum. In addition, this study indicates that thalamic delivery of AAV vectors should be combined with additional targets to supply the cerebellum and spinal cord with therapeutic levels of enzyme necessary to achieve complete correction of the neurological phenotype in GM1 mice.

Sialidases in vertebrates: a family of enzymes tailored for several cell functions.

This review summarizes the recent research development on vertebrate sialidase biology. Sialic acid-containing compounds play important roles in many physiological processes, including cell proliferation, apoptosis and differentiation, control of cell adhesion, immune surveillance, and clearance of plasma proteins. In this context, sialidases, the glycohydrolases that remove the terminal sialic acid at the non-reducing end of various glycoconjugates, perform an equally pivotal function. Sialidases in higher organisms are differentially expressed in cells and tissues/organs, with particular subcellular distribution and substrate specificity: they are the lysosomal (NEU1), the cytosolic (NEU2), and plasma membrane- and intracellular-associated sialidases (NEU3 and NEU4). The molecular cloning of several mammalian sialidases since 1993 has boosted research in this field. Here we summarize the results obtained since 2002, when the last general review on the molecular biology of mammalian sialidases was written. In those few years many original papers dealing with different aspects of sialidase biology have been published, highlighting the increasing relevance of these enzymes in glycobiology. Attention has also been paid to the trans-sialidases, which transfer sialic acid residues from a donor sialoconjugate to an acceptor asialo substrate. These enzymes are abundantly distributed in trypanosomes and employed to express pathogenicity, also in humans. There are structural similarities and strategic differences at the level of the active site between the mammalian sialidases and trans-sialidases. A better knowledge of these properties may permit the design of better anti-pathogen drugs.