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Ninu (greater bilby, Macrotis lagotis) are desert-dwelling, culturally and ecologically important marsupials. In collaboration with Indigenous rangers and conservation managers, we generated the Ninu chromosome-level genome assembly (3.66 Gbp) and genome sequences for the extinct Yallara (lesser bilby, Macrotis leucura). We developed and tested a scat single-nucleotide polymorphism panel to inform current and future conservation actions, undertake ecological assessments and improve our understanding of Ninu genetic diversity in managed and wild populations. We also assessed the beneficial impact of translocations in the metapopulation (N = 363 Ninu). Resequenced genomes (temperate Ninu, 6; semi-arid Ninu, 6; and Yallara, 4) revealed two major population crashes during global cooling events for both species and differences in Ninu genes involved in anatomical and metabolic pathways. Despite their 45-year captive history, Ninu have fewer long runs of homozygosity than other larger mammals, which may be attributable to their boom-bust life history. Here we investigated the unique Ninu biology using 12 tissue transcriptomes revealing expression of all 115 conserved eutherian chorioallantoic placentation genes in the uterus, an XY1Y2 sex chromosome system and olfactory receptor gene expansions. Together, we demonstrate the holistic value of genomics in improving key conservation actions, understanding unique biological traits and developing tools for Indigenous rangers to monitor remote wild populations.
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Conservación de los Recursos Naturales , Genoma , Marsupiales , Animales , Marsupiales/genética , Australia , Polimorfismo de Nucleótido Simple , Extinción BiológicaRESUMEN
Chemical cues in subterranean habitats differ highly from those on the surface due to the contrasting environmental conditions, such as absolute darkness, high humidity or food scarcity. Subterranean animals underwent changes to their sensory systems to facilitate the perception of essential stimuli for underground lifestyles. Despite representing unique systems to understand biological adaptation, the genomic basis of chemosensation across cave-dwelling species remains unexplored from a macroevolutionary perspective. Here, we explore the evolution of chemoreception in three beetle tribes that underwent at least six independent transitions to the underground, through a phylogenomics spyglass. Our findings suggest that the chemosensory gene repertoire varies dramatically between species. Overall, no parallel changes in the net rate of evolution of chemosensory gene families were detected prior, during, or after the habitat shift among subterranean lineages. Contrarily, we found evidence of lineage-specific changes within surface and subterranean lineages. However, our results reveal key duplications and losses shared between some of the lineages transitioning to the underground, including the loss of sugar receptors and gene duplications of the highly conserved ionotropic receptors IR25a and IR8a, involved in thermal and humidity sensing among other olfactory roles in insects. These duplications were detected both in independent subterranean lineages and their surface relatives, suggesting parallel evolution of these genes across lineages giving rise to cave-dwelling species. Overall, our results shed light on the genomic basis of chemoreception in subterranean beetles and contribute to our understanding of the genomic underpinnings of adaptation to the subterranean lifestyle at a macroevolutionary scale.
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Escarabajos , Animales , Escarabajos/genética , Filogenia , Ecosistema , Insectos , CuevasRESUMEN
Adaptation to life in caves is often accompanied by dramatically convergent changes across distantly related taxa, epitomized by the loss or reduction of eyes and pigmentation. Nevertheless, the genomic underpinnings underlying cave-related phenotypes are largely unexplored from a macroevolutionary perspective. Here we investigate genome-wide gene evolutionary dynamics in three distantly related beetle tribes with at least six instances of independent colonization of subterranean habitats, inhabiting both aquatic and terrestrial underground systems. Our results indicate that remarkable gene repertoire changes mainly driven by gene family expansions occurred prior to underground colonization in the three tribes, suggesting that genomic exaptation may have facilitated a strict subterranean lifestyle parallelly across beetle lineages. The three tribes experienced both parallel and convergent changes in the evolutionary dynamics of their gene repertoires. These findings pave the way towards a deeper understanding of the evolution of the genomic toolkit in hypogean fauna.
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Escarabajos , Genómica , Animales , Aclimatación , Cuevas , Escarabajos/genética , Evolución MolecularRESUMEN
Subterranean habitats are generally very stable environments, and as such evolutionary transitions of organisms from surface to subterranean lifestyles may cause considerable shifts in physiology, particularly with respect to thermal tolerance. In this study we compared responses to heat shock at the molecular level in a geographically widespread, surface-dwelling water beetle to a congeneric subterranean species restricted to a single aquifer (Dytiscidae: Hydroporinae). The obligate subterranean beetle Paroster macrosturtensis is known to have a lower thermal tolerance compared to surface lineages (CTmax 38 °C cf. 42-46 °C), but the genetic basis of this physiological difference has not been characterized. We experimentally manipulated the thermal environment of 24 individuals to demonstrate that both species can mount a heat shock response at high temperatures (35 °C), as determined by comparative transcriptomics. However, genes involved in these responses differ between species and a far greater number were differentially expressed in the surface taxon, suggesting it can mount a more robust heat shock response; these data may underpin its higher thermal tolerance compared to subterranean relatives. In contrast, the subterranean species examined not only differentially expressed fewer genes in response to increasing temperatures, but also in the presence of the experimental setup employed here alone. Our results suggest P. macrosturtensis may be comparatively poorly equipped to respond to both thermally induced stress and environmental disturbances more broadly. The molecular findings presented here have conservation implications for P. macrosturtensis and contribute to a growing narrative concerning weakened thermal tolerances in obligate subterranean organisms at the molecular level.
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Escarabajos , Animales , Escarabajos/genética , Ecosistema , Respuesta al Choque Térmico/genética , TranscriptomaRESUMEN
Two new species, Abantiades concordia sp. nov. and Abantiades malleus sp. nov., are described from Australia. Both species were collected in the Eastern Goldfields subregion of the Coolgardie bioregion in Western Australia. Abantiades concordia sp. nov. is shown to be closely related to A. paradoxa (Tindale, 1932) by sequence similarity of the mtDNA (COI) gene. The female of A. paradoxa is also described here for the first time. Abantiades paradoxa and the new species A. concordia sp. nov. are morphologically similar with respect to the structure of their genitalia, sternite VIII, wing patterning and their antennae with bi-forked rami. Abantiades malleus sp. nov. is quite distinct by sequence similarity of the mtDNA (COI) gene, but related in a clade with A. marcidus Tindale,1932, A. albofasciatus (Swinhoe, 1892), and A. furva (Tindale,1932), the latter species once placed in the synonymised Bordaia Tindale, 1932. Discussion of similar species once grouped under the genus Bordaia and under the genus Trictena Meyrick, 1890 (both junior synonyms of Abantiades Herrich-Schffer, 1855) is also included.
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Mariposas Nocturnas , Animales , Australia , ADN Mitocondrial/genética , Femenino , Genitales , Mariposas Nocturnas/genética , Alas de AnimalesRESUMEN
Mitochondrial heteroplasmy is the occurrence of more than one type of mitochondrial DNA within a single individual. Although generally reported to occur in a small subset of individuals within a species, there are some instances of widespread heteroplasmy across entire populations. Amphylaeus morosus is an Australian native bee species in the diverse and cosmopolitan bee family Colletidae. This species has an extensive geographical range along the eastern Australian coast, from southern Queensland to western Victoria, covering approximately 2,000 km. Seventy individuals were collected from five localities across this geographical range and sequenced using Sanger sequencing for the mitochondrial cytochrome c oxidase subunit I (COI) gene. These data indicate that every individual had the same consistent heteroplasmic sites but no other nucleotide variation, suggesting two conserved and widespread heteroplasmic mitogenomes. Ion Torrent shotgun sequencing revealed that heteroplasmy occurred across multiple mitochondrial protein-coding genes and is unlikely explained by transposition of mitochondrial genes into the nuclear genome (NUMTs). DNA sequence data also demonstrated a consistent co-infection of Wolbachia across the A. morosus distribution with every individual infected with both bacterial strains. Our data are consistent with the presence of two mitogenomes within all individuals examined in this species and suggest a major divergence from standard patterns of mitochondrial inheritance. Because the host's mitogenome and the Wolbachia genome are genetically linked through maternal inheritance, we propose three possible hypotheses that could explain maintenance of the widespread and conserved co-occurring bacterial and mitochondrial genomes in this species.
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In the framework of neutral theory of molecular evolution, genes specific to the development and function of eyes in subterranean animals living in permanent darkness are expected to evolve by relaxed selection, ultimately becoming pseudogenes. However, definitive empirical evidence for the role of neutral processes in the loss of vision over evolutionary time remains controversial. In previous studies, we characterized an assemblage of independently-evolved water beetle (Dytiscidae) species from a subterranean archipelago in Western Australia, where parallel vision and eye loss have occurred. Using a combination of transcriptomics and exon capture, we present evidence of parallel coding sequence decay, resulting from the accumulation of frameshift mutations and premature stop codons, in eight phototransduction genes (arrestins, opsins, ninaC and transient receptor potential channel genes) in 32 subterranean species in contrast to surface species, where these genes have open reading frames. Our results provide strong evidence to support neutral evolutionary processes as a major contributing factor to the loss of phototransduction genes in subterranean animals, with the ultimate fate being the irreversible loss of a light detection system.
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Escarabajos , Animales , Escarabajos/genética , Evolución Molecular , Opsinas/genética , Filogenia , AguaRESUMEN
Most subterranean animals are assumed to have evolved from surface ancestors following colonization of a cave system; however, very few studies have raised the possibility of "subterranean speciation" in underground habitats (i.e., obligate cave-dwelling organisms [troglobionts] descended from troglobiotic ancestors). Numerous endemic subterranean diving beetle species from spatially discrete calcrete aquifers in Western Australia (stygobionts) have evolved independently from surface ancestors; however, several cases of sympatric sister species raise the possibility of subterranean speciation. We tested this hypothesis using vision (phototransduction) genes that are evolving under neutral processes in subterranean species and purifying selection in surface species. Using sequence data from 32 subterranean and five surface species in the genus Paroster (Dytiscidae), we identified deleterious mutations in long wavelength opsin (lwop), arrestin 1 (arr1), and arrestin 2 (arr2) shared by a sympatric sister-species triplet, arr1 shared by a sympatric sister-species pair, and lwop and arr2 shared among closely related species in adjacent calcrete aquifers. In all cases, a common ancestor possessed the function-altering mutations, implying they were already adapted to aphotic environments. Our study represents one of the first confirmed cases of subterranean speciation in cave insects. The assessment of genes undergoing pseudogenization provides a novel way of testing modes of speciation and the history of diversification in blind cave animals.
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Escarabajos/genética , Flujo Genético , Especiación Genética , Proteínas de Insectos/genética , Visión Ocular/genética , Animales , Arrestinas/genética , Agua Subterránea , Opsinas/genéticaRESUMEN
BACKGROUND: Repetitive DNA sequences, including transposable elements (TEs) and tandemly repeated satellite DNA (satDNAs), collectively called the "repeatome", are found in high proportion in organisms across the Tree of Life. Grasshoppers have large genomes, averaging 9 Gb, that contain a high proportion of repetitive DNA, which has hampered progress in assembling reference genomes. Here we combined linked-read genomics with transcriptomics to assemble, characterize, and compare the structure of repetitive DNA sequences in four chromosomal races of the morabine grasshopper Vandiemenella viatica species complex and determine their contribution to genome evolution. RESULTS: We obtained linked-read genome assemblies of 2.73-3.27 Gb from estimated genome sizes of 4.26-5.07 Gb DNA per haploid genome of the four chromosomal races of V. viatica. These constitute the third largest insect genomes assembled so far. Combining complementary annotation tools and manual curation, we found a large diversity of TEs and satDNAs, constituting 66 to 75% per genome assembly. A comparison of sequence divergence within the TE classes revealed massive accumulation of recent TEs in all four races (314-463 Mb per assembly), indicating that their large genome sizes are likely due to similar rates of TE accumulation. Transcriptome sequencing showed more biased TE expression in reproductive tissues than somatic tissues, implying permissive transcription in gametogenesis. Out of 129 satDNA families, 102 satDNA families were shared among the four chromosomal races, which likely represent a diversity of satDNA families in the ancestor of the V. viatica chromosomal races. Notably, 50 of these shared satDNA families underwent differential proliferation since the recent diversification of the V. viatica species complex. CONCLUSION: This in-depth annotation of the repeatome in morabine grasshoppers provided new insights into the genome evolution of Orthoptera. Our TEs analysis revealed a massive recent accumulation of TEs equivalent to the size of entire Drosophila genomes, which likely explains the large genome sizes in grasshoppers. Despite an overall high similarity of the TE and satDNA diversity between races, the patterns of TE expression and satDNA proliferation suggest rapid evolution of grasshopper genomes on recent timescales.
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Elementos Transponibles de ADN/genética , ADN Satélite/genética , Genoma de los Insectos , Animales , Femenino , Saltamontes/genética , MasculinoAsunto(s)
Estructuras Genéticas , Genética de Población , Humanos , Selección Genética , Australia del SurRESUMEN
The origin of the mammalian order Eulipotyphla has been debated intensively with arguments around whether they began diversifying before or after the Cretaceous-Palaeogene (K-Pg) boundary at 66â¯Ma. Here, we used an in-solution nucleotide capture method and next generation DNA sequencing to determine the sequence of hundreds of ultra-conserved elements (UCEs), and conducted phylogenomic and molecular dating analyses for the four extant eulipotyphlan lineages-Erinaceidae, Solenodontidae, Soricidae, and Talpidae. Concatenated maximum-likelihood analyses with single or partitioned models and a coalescent species-tree analysis showed that divergences among the four major eulipotyphlan lineages occurred within a short period of evolutionary time, but did not resolve the interrelationships among them. Alternative suboptimal phylogenetic hypotheses received consistently the same amount of support from different UCE loci, and were not significantly different from the maximum likelihood tree topology, suggesting the prevalence of stochastic lineage sorting. Molecular dating analyses that incorporated among-lineage evolutionary rate differences supported a scenario where the four eulipotyphlan families diversified between 57.8 and 63.2â¯Ma. Given short branch lengths with low support values, traces of rampant genome-wide stochastic lineage sorting, and post K-Pg diversification, we concluded that the crown eulipotyphlan lineages arose through a rapid diversification after the K-Pg boundary when novel niches were created by the mass extinction of species.
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Secuencia Conservada , Mamíferos/clasificación , Mamíferos/genética , Filogenia , Animales , Composición de Base/genética , Calibración , Secuencia Conservada/genética , Variación Genética , Funciones de Verosimilitud , Factores de TiempoRESUMEN
The Microgastrinae are a hugely diverse subfamily of endoparasitoid wasps of lepidopteran caterpillars. They are important in agriculture as biological control agents and play a significant ecological role in the regulation of caterpillar populations. Whilst the group has been the focus of intensive rearing and DNA barcoding studies in the Northern Hemisphere, the Australian fauna has received little attention. In total, 99 species have been described from or have been introduced into Australia, but the real species diversity for the region is clearly much larger than this. In this study, museum ethanol samples and recent field collections were mined for hundreds of specimens of microgastrine wasps, which were then barcoded for the COI region, ITS2 ribosomal spacer and the wingless nuclear genes, using a pooled sequencing approach on an Illumina Miseq system. Full COI sequences were obtained for 525 individuals which, when combined with 162 publicly available sequences, represented 417 haplotypes, and a total of 236 species were delimited using a consensus approach. By more than doubling the number of known microgastrine wasp species in Australia, our study highlights the value of DNA barcoding in the context of employing high-throughput sequencing methods of bulk ethanol museum collections for biodiversity assessment.
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Characterisation of squamate major histocompatibility complex (MHC) genes has lagged behind other taxonomic groups. MHC genes encode cell-surface glycoproteins that present self- and pathogen-derived peptides to T cells and play a critical role in pathogen recognition. Here we characterise MHC class I transcripts for an agamid lizard (Ctenophorus decresii) and investigate the evolution of MHC class I in Iguanian lizards. An iterative assembly strategy was used to identify six full-length C. decresii MHC class I transcripts, which were validated as likely to encode classical class I MHC molecules. Evidence for exon shuffling recombination was uncovered for C. decresii transcripts and Bayesian phylogenetic analysis of Iguanian MHC class I sequences revealed a pattern expected under a birth-and-death mode of evolution. This work provides a stepping stone towards further research on the agamid MHC class I region.
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Antígenos de Histocompatibilidad Clase I/genética , Lagartos/genética , Complejo Mayor de Histocompatibilidad/genética , Animales , Australia , Teorema de Bayes , Evolución Biológica , Evolución Molecular , Exones/genética , Lagartos/inmunología , Filogenia , Recombinación Genética , TranscriptomaRESUMEN
Three new species of Koonunga were discovered in surface and subterranean waters in southern Australia, and were defined using mtDNA analyses and morphology. The new species are: Koonunga hornei Leijs & King; K. tatiaraensis Leijs & King and K. allambiensis Leijs & King. Molecular clock analyses indicate that the divergence times of the species are older than the landscape that they currently inhabit. Different scenarios explaining this apparent discrepancy are discussed in the context of the palaeography of the area. A freshwater epigean origin for Koonunga is considered the most likely hypothesis, whereby some lineages made the transition to the subterranean environment within the last few million years influenced by significant climatic cooling/drying. We discuss the possibility that one stygobitic lineage secondarily regained some of its body pigmentation as adaptation to increased photic conditions after cave collapse and forming of cenotes during the last glacial maximum.
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Crustáceos/fisiología , Evolución Molecular , Animales , Australia , Ecosistema , Geografía , FilogeniaRESUMEN
The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. FcγRIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of FcγRIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of FcγRIIa (FcγRIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence FcγRIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for FcγRIIa (IV.3), FcγRIIb (X63-21), and a pan FcγRII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of FcγRIIa and FcγRIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of FcγRIIa-HR binds Ag-Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.
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Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/inmunología , Inmunoglobulina G/inmunología , Receptores de IgG/química , Receptores de IgG/inmunología , Animales , Complejo Antígeno-Anticuerpo/genética , Cristalografía por Rayos X , Mapeo Epitopo , Humanos , Inmunoglobulina G/química , Ratones , Modelos Moleculares , Polimorfismo de Nucleótido Simple , Estructura Cuaternaria de Proteína , Receptores de IgG/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Transducción de Señal/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
The aggregation of cell surface FcRs by immune complexes induces a number of important Ab-dependent effector functions. However, despite numerous studies that examine receptor function, very little is known about the molecular organization of these receptors within the cell. In this study, protein complementation, mutagenesis, and ligand binding analyses demonstrate that human FcgammaRIIa is present as a noncovalent dimer form. Protein complementation studies found that FcgammaRIIa molecules are closely associated. Mutagenesis of the dimer interface, as identified by crystallographic analyses, did not affect ligand binding yet caused significant alteration to the magnitude and kinetics of receptor phosphorylation. The data suggest that the ligand binding and the dimer interface are distinct regions within the receptor, and noncovalent dimerization of FcgammaRIIa may be an essential feature of the FcgammaRIIa signaling cascade.
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Antígenos CD/genética , Antígenos CD/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Animales , Antígenos CD/fisiología , Sitios de Unión/genética , Sitios de Unión/inmunología , Células CHO , Cricetinae , Cricetulus , Dimerización , Regulación hacia Abajo/genética , Humanos , Inmunoglobulina G/metabolismo , Ligandos , Metotrexato/metabolismo , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Prolina/genética , Receptores de IgG/fisiología , Serina/genética , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
Antibodies targeting human epithelial carcinomas bearing Lewis Y (Le(y)) carbohydrate antigens provide a striking illustration of convergent immune recognition. We report a 1.9A resolution crystal structure of the Fab of a humanized antibody (hu3S193) in complex with the Le(y) tetrasaccharide, Fuc(alpha 1-->2)Gal(beta 1-->4)[Fuc(alpha 1-->3)]GlcNAc. Comparisons of the hu3S193 and BR96 antibodies bound to Le(y) tumor antigens revealed extremely similar mechanisms for recognition of the carbohydrate epitopes. Solvent plays a critical role in hu3S193 antibody binding to the Le(y) carbohydrate epitope. Specificity for Le(y) is maintained because a conserved pocket accepts an N-acetyl group of the core Gal(beta 1-->4)GlcNAc disaccharide. Closely related blood-group determinants (Le(a) and Le(b)) cannot enter the specificity pocket, making the Le(y) antibodies promising candidates for immunotherapy of epithelial cancer.
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Anticuerpos Antineoplásicos/inmunología , Antígenos de Carbohidratos Asociados a Tumores/química , Carbohidratos/inmunología , Antígenos del Grupo Sanguíneo de Lewis/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Sitios de Unión de Anticuerpos , Conformación de Carbohidratos , Secuencia de Carbohidratos , Carcinoma/inmunología , Regiones Determinantes de Complementariedad , Cristalografía por Rayos X , Epítopos , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Antígenos del Grupo Sanguíneo de Lewis/inmunología , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasias Glandulares y Epiteliales/inmunología , Oligosacáridos/química , Oligosacáridos/inmunologíaRESUMEN
The lysosomal hydrolase N-acetylgalactosamine 4-sulfatase (4-sulfatase) is required for the degradation of the glycosaminoglycan substrates dermatan and chondroitin sulfate. A 4-sulfatase deficiency results in the accumulation of undegraded substrate and causes the severe lysosomal storage disorder mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome. A wide variation in clinical severity is observed between MPS VI patients and reflects the number of different 4-sulfatase mutations that can cause the disorder. The most common 4-sulfatase mutation, Y210C, was detected in approximately 10% of MPS VI patients and has been associated with an attenuated clinical phenotype when compared to the archetypical form of MPS VI. To define the molecular defect caused by this mutation, Y210C 4-sulfatase was expressed in Chinese hamster ovary (CHO-K1) cells for protein and cell biological analysis. Biosynthetic studies revealed that Y210C 4-sulfatase was synthesized at a comparable molecular size and amount to wild-type 4-sulfatase, but there was evidence of delayed processing, traffic, and stability of the mutant protein. Thirty-three percent of the intracellular Y210C 4-sulfatase remained as a precursor form, for at least 8 h post labeling and was not processed to the mature lysosomal form. However, unlike other 4-sulfatase mutations causing MPS VI, a significant amount of Y210C 4-sulfatase escaped the endoplasmic reticulum and was either secreted from the expression cells or underwent delayed intracellular traffic. Sixty-seven percent of the intracellular Y210C 4-sulfatase was processed to the mature form (43, 8, and 7 kDa molecular mass forms) by a proteolytic processing step known to occur in endosomes-lysosomes. Treatment of Y210C CHO-K1 cells with the protein stabilizer glycerol resulted in increased amounts of Y210C 4-sulfatase in endosomes, which was eventually trafficked to the lysosome after a long, 24 h chase time. This demonstrated delayed traffic of Y210C 4-sulfatase to the lysosomal compartment. The endosomal Y210C 4-sulfatase had a low specific activity, suggesting that the mutant protein also had problems with stability. Treatment of Y210C CHO-K1 cells with the protease inhibitor ALLM resulted in an increased amount of mature Y210C 4-sulfatase localized in lysosomes, but this protein had a very low level of activity. This indicated that the mutant protein was being inactivated and degraded at an enhanced rate in the lysosomal compartment. Biochemical analysis of Y210C 4-sulfatase revealed a normal pH optimum for the mutant protein but demonstrated a reduced enzyme activity with time, also consistent with a protein stability problem. This study indicated that multiple subcellular and biochemical processes can contribute to the biogenesis of mutant protein and may in turn influence the clinical phenotype of a patient. In MPS VI patients with a Y210C allele, the composite effect of different stages of intracellular processing/handling and environment has been shown to cause a reduced level of Y210C 4-sulfatase protein and activity, resulting in an attenuated clinical phenotype.