A mutation in SLC37A4 causes a dominantly inherited congenital disorder of glycosylation characterized by liver dysfunction.

SLC37A4 encodes an endoplasmic reticulum (ER)-localized multitransmembrane protein required for transporting glucose-6-phosphate (Glc-6P) into the ER. Once transported into the ER, Glc-6P is subsequently hydrolyzed by tissue-specific phosphatases to glucose and inorganic phosphate during times of glucose depletion. Pathogenic variants in SLC37A4 cause an established recessive disorder known as glycogen storage disorder 1b characterized by liver and kidney dysfunction with neutropenia. We report seven individuals who presented with liver dysfunction multifactorial coagulation deficiency and cardiac issues and were heterozygous for the same variant, c.1267C>T (p.Arg423∗), in SLC37A4; the affected individuals were from four unrelated families. Serum samples from affected individuals showed profound accumulation of both high mannose and hybrid type N-glycans, while N-glycans in fibroblasts and undifferentiated iPSC were normal. Due to the liver-specific nature of this disorder, we generated a CRISPR base-edited hepatoma cell line harboring the c.1267C>T (p.Arg423∗) variant. These cells replicated the secreted abnormalities seen in serum N-glycosylation, and a portion of the mutant protein appears to relocate to a distinct, non-Golgi compartment, possibly ER exit sites. These cells also show a gene dosage-dependent alteration in the Golgi morphology and reduced intraluminal pH that may account for the altered glycosylation. In summary, we identify a recurrent mutation in SLC37A4 that causes a dominantly inherited congenital disorder of glycosylation characterized by coagulopathy and liver dysfunction with abnormal serum N-glycans.

Biallelic missense variants in COG3 cause a congenital disorder of glycosylation with impairment of retrograde vesicular trafficking.

Biallelic variants in genes for seven out of eight subunits of the conserved oligomeric Golgi complex (COG) are known to cause recessive congenital disorders of glycosylation (CDG) with variable clinical manifestations. COG3 encodes a constituent subunit of the COG complex that has not been associated with disease traits in humans. Herein, we report two COG3 homozygous missense variants in four individuals from two unrelated consanguineous families that co-segregated with COG3-CDG presentations. Clinical phenotypes of affected individuals include global developmental delay, severe intellectual disability, microcephaly, epilepsy, facial dysmorphism, and variable neurological findings. Biochemical analysis of serum transferrin from one family showed the loss of a single sialic acid. Western blotting on patient-derived fibroblasts revealed reduced COG3 and COG4. Further experiments showed delayed retrograde vesicular recycling in patient cells. This report adds to the knowledge of the COG-CDG network by providing collective evidence for a COG3-CDG rare disease trait and implicating a likely pathology of the disorder as the perturbation of Golgi trafficking.

A Recurrent De Novo Heterozygous COG4 Substitution Leads to Saul-Wilson Syndrome, Disrupted Vesicular Trafficking, and Altered Proteoglycan Glycosylation.

The conserved oligomeric Golgi (COG) complex is involved in intracellular vesicular transport, and is composed of eight subunits distributed in two lobes, lobe A (COG1-4) and lobe B (COG5-8). We describe fourteen individuals with Saul-Wilson syndrome, a rare form of primordial dwarfism with characteristic facial and radiographic features. All affected subjects harbored heterozygous de novo variants in COG4, giving rise to the same recurrent amino acid substitution (p.Gly516Arg). Affected individuals' fibroblasts, whose COG4 mRNA and protein were not decreased, exhibited delayed anterograde vesicular trafficking from the ER to the Golgi and accelerated retrograde vesicular recycling from the Golgi to the ER. This altered steady-state equilibrium led to a decrease in Golgi volume, as well as morphologic abnormalities with collapse of the Golgi stacks. Despite these abnormalities of the Golgi apparatus, protein glycosylation in sera and fibroblasts from affected subjects was not notably altered, but decorin, a proteoglycan secreted into the extracellular matrix, showed altered Golgi-dependent glycosylation. In summary, we define a specific heterozygous COG4 substitution as the molecular basis of Saul-Wilson syndrome, a rare skeletal dysplasia distinct from biallelic COG4-CDG.

Defining the clinical phenotype of Saul-Wilson syndrome.

PURPOSE: Four patients with Saul-Wilson syndrome were reported between 1982 and 1994, but no additional individuals were described until 2018, when the molecular etiology of the disease was elucidated. Hence, the clinical phenotype of the disease remains poorly defined. We address this shortcoming by providing a detailed characterization of its phenotype. METHODS: Retrospective chart reviews were performed and primary radiographs assessed for all 14 individuals. Four individuals underwent detailed ophthalmologic examination by the same physician. Two individuals underwent gynecologic evaluation. Z-scores for height, weight, head circumference and body mass index were calculated at different ages. RESULTS: All patients exhibited short stature, with sharp decline from the mean within the first months of life, and a final height Z-score between -4 and -8.5 standard deviations. The facial and radiographic features evolved over time. Intermittent neutropenia was frequently observed. Novel findings included elevation of liver transaminases, skeletal fragility, rod-cone dystrophy, and cystic macular changes. CONCLUSIONS: Saul-Wilson syndrome presents a remarkably uniform phenotype, and the comprehensive description of our cohort allows for improved understanding of the long-term morbidity of the condition, establishment of follow-up recommendations for affected individuals, and documentation of the natural history into adulthood for comparison with treated patients, when therapeutics become available.

Functional regions of the peroxin Pex19 necessary for peroxisome biogenesis.

The peroxins Pex19 and Pex3 play an indispensable role in peroxisomal membrane protein (PMP) biogenesis, peroxisome division, and inheritance. Pex19 plays multiple roles in these processes, but how these functions relate to the structural organization of the Pex19 domains is unresolved. To this end, using deletion mutants, we mapped the Pex19 regions required for peroxisome biogenesis in the yeast Pichia pastoris Surprisingly, import-competent peroxisomes still formed when Pex19 domains previously believed to be required for biogenesis were deleted, although the peroxisome size was larger than that in wild-type cells. Moreover, these mutants exhibited a delay of 14-24 h in peroxisome biogenesis. The shortest functional N-terminal (NTCs) and C-terminal constructs (CTCs) were Pex19 (aa 1-150) and Pex19 (aa 89-300), respectively. Deletions of the N-terminal Pex3-binding site disrupted the direct interactions of Pex19 with Pex3, but preserved interactions with a membrane peroxisomal targeting signal (mPTS)-containing PMP, Pex10. In contrast, deletion of the C-terminal mPTS-binding domain of Pex19 disrupted its interaction with Pex10 while leaving the Pex19-Pex3 interactions intact. However, Pex11 and Pex25 retained their interactions with both N- and C-terminal deletion mutants. NTC-CTC co-expression improved growth and reversed the larger-than-normal peroxisome size observed with the single deletions. Pex25 was critical for peroxisome formation with the CTC variants, and its overexpression enhanced their interactions with Pex3 and aided the growth of both NTC and CTC Pex19 variants. In conclusion, physical segregation of the Pex3- and PMP-binding domains of Pex19 has provided novel insights into the modular architecture of Pex19. We define the minimum region of Pex19 required for peroxisome biogenesis and a unique role for Pex25 in this process.

Salinicola tamaricis sp. nov., a heavy-metal-tolerant, endophytic bacterium isolated from the halophyte Tamarix chinensis Lour.

A Gram-stain-negative, rod-shaped bacterium, strain F01T, was isolated from leaves of Tamarix chinensis Lour. The isolate grew optimally at 30 °C, at pH 7.0 and with 5.0 % (w/v) NaCl, and showed a high tolerance to manganese, lead, nickel, ferrous ions and copper ions. The major fatty acids were C18 : 1ω7c and C16 : 0, and the predominant respiratory quinone was Q-9. Polar lipids were dominated by diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, unidentified aminoglycolipids and phospholipids. The DNA G+C content was 65.8 %. Based on multilocus phylogenetic analysis, strain F01T belonged to the genus Salinicola, with highest 16S rRNA gene sequence similarity to Salinicola peritrichatus CGMCC 1.12381T (97.7 %). The level of DNA-DNA hybridization between strain F01T and closely related Salinicola strains was well below 70 %. According to the phenotypic, genetic and chemotaxonomic data, strain F01T is considered to represent a novel species in the genus Salinicola, for which the name Salinicola tamaricis sp. nov. is proposed. The type strain is F01T (=CCTCC AB 2015304T=KCTC 42855T).

Larsenimonas suaedae sp. nov., a moderately halophilic, endophytic bacterium isolated from the halophyte Suaeda salsa.

A moderately halophilic, Gram-stain-negative, non-endospore-forming endophytic bacterium designated strain ST307T was isolated from the euhalophyte Suaeda salsa in Dongying, China. Strain ST307T was aerobic, rod-shaped, motile and orange-yellow-pigmented. The organism grew at NaCl concentrations of 0.6-20 % (w/v) (optimum 5-6 %, w/v), at temperatures of 5-45 °C (optimum 35 °C) and at pH 5-9 (optimum pH 7-8). It accumulated poly-ß-hydroxybutyric acid and produced exopolysaccharides. The major fatty acids were C18 : 1ω7c/C18 : 1ω6c, C16 : 0 and C16 : 1ω7c/C16 : 1ω6c. The predominant lipoquinone was ubiquinone Q-9. The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, a glycoaminolipid and a phosphoglycoaminolipid. The DNA G+C content was 60.5 mol%. Phylogenetic analyses of 16S rRNA gene sequences and concatenated atpA, rpoD and secA gene sequences revealed that the strain represents a member of the genus Larsenimonas. The closest related type strain was Larsenimonas salina M1-18T. Mean DNA-DNA relatedness values between strain ST307T and the related species L. salina M1-18T, Chromohalobacter beijerinckii DSM 7218T, C. canadensis DSM 6769T, C. israelensis DSM 6768T, C. marismortui CGMCC 1.2321T, C. nigrandesensis DSM 14323T, C. salexigens DSM 3043T and C. sarecensis DSM 15547T were 15±2-45±1 %. On the basis of phenotypic, chemotaxonomic and molecular features, strain ST307T clearly represents a novel species of the genus Larsenimonas. The name Larsenimonassuaedae sp. nov. is proposed, with ST307T (=CGMCC 1.8902T=DSM 22428T) as the type strain.

The Swedish COG6-CDG experience and a comprehensive literature review.

Here, we present the first two Swedish cases of Conserved Oligomeric Golgi complex subunit 6-congenital disorders of glycosylation (COG6-CDG). Their clinical symptoms include intellectual disability, Attention Deficit/Hyperactivity Disorder (ADHD), delayed brain myelinization, progressive microcephaly, joint laxity, hyperkeratosis, frequent infections, and enamel hypoplasia. In one family, compound heterozygous variants in COG6 were identified, where one (c.785A>G; p.Tyr262Cys) has previously been described in patients of Moroccan descent, whereas the other (c.238G>A; p.Glu80Lys) is undescribed. On the other hand, a previously undescribed homozygous duplication (c.1793_1795dup) was deemed the cause of the disease. To confirm the pathogenicity of the variants, we treated patient and control fibroblasts with the ER-Golgi transport inhibitor Brefeldin-A and show that patient cells manifest a significantly slower anterograde and retrograde ER-Golgi transport.

[Analysis of the risk factors related to prognosis of severe intra-abdominal infection in surgical intensive care unit].

OBJECTIVE: To investigate the risk factors related to prognosis of patients with severe intra-abdominal infection (SIAI) in surgical intensive care unit (SICU). METHODS: Clinical data of 69 patients with SIAI, who were hospitalized during January 2008 and April 2011 in SICU, were analyzed retrospectively. According to the outcome at discharge from SICU, 69 patients were divided into two groups: the survivors 42 cases; the deceased 27 cases. The potential risk factors of SIAI patients in SICU were analyzed by univariate analysis and logistic regression analysis. RESULTS: Univariate analysis showed that average acute physiology and chronic health evaluation II (APACHEII) scores (the survival group: 11.76 ± 3.48; the death group: 17.12 ± 4.50), inadequate treatment of intra-abdominal infection (3 survivors, 21 deceased), inadequate initial antibiotic treatment (3 survivors, 15 deceased) and septic shock (24 survivors, 27 deceased) were risk factors for mortality (all P < 0.01). Logistic regression analysis showed that average APACHEII scores over 15 [relative risk (RR): 6.846, P value: 0.044] and insufficient treatment of intra-abdominal infection (RR: 21.319, P value: 0.018) were the independent risk factors in SIAI patients in SICU. CONCLUSION: To decrease the mortality of SIAI patients in SICU, more attention should be paid to monitor APACHEII scores and to handle intra-abdominal infection adequately.

COG4 mutation in Saul-Wilson syndrome selectively affects secretion of proteins involved in chondrogenesis in chondrocyte-like cells.

Saul-Wilson syndrome is a rare skeletal dysplasia caused by a heterozygous mutation in COG4 (p.G516R). Our previous study showed that this mutation affected glycosylation of proteoglycans and disturbed chondrocyte elongation and intercalation in zebrafish embryos expressing the COG4p.G516R variant. How this mutation causes chondrocyte deficiencies remain unsolved. To analyze a disease-relevant cell type, COG4p.G516R variant was generated by CRISPR knock-in technique in the chondrosarcoma cell line SW1353 to study chondrocyte differentiation and protein secretion. COG4p.G516R cells display impaired protein trafficking and altered COG complex size, similar to SWS-derived fibroblasts. Both SW1353 and HEK293T cells carrying COG4p.G516R showed very modest, cell-type dependent changes in N-glycans. Using 3D culture methods, we found that cells carrying the COG4p.G516R variant made smaller spheroids and had increased apoptosis, indicating impaired in vitro chondrogenesis. Adding WT cells or their conditioned medium reduced cell death and increased spheroid sizes of COG4p.G516R mutant cells, suggesting a deficiency in secreted matrix components. Mass spectrometry-based secretome analysis showed selectively impaired protein secretion, including MMP13 and IGFBP7 which are involved in chondrogenesis and osteogenesis. We verified reduced expression of chondrogenic differentiation markers, MMP13 and COL10A1 and delayed response to BMP2 in COG4p.G516R mutant cells. Collectively, our results show that the Saul-Wilson syndrome COG4p.G516R variant selectively affects the secretion of multiple proteins, especially in chondrocyte-like cells which could further cause pleiotropic defects including hampering long bone growth in SWS individuals.

Origin of cytoplasmic GDP-fucose determines its contribution to glycosylation reactions.

Biosynthesis of macromolecules requires precursors such as sugars or amino acids, originating from exogenous/dietary sources, reutilization/salvage of degraded molecules, or de novo synthesis. Since these sources are assumed to contribute to one homogenous pool, their individual contributions are often overlooked. Protein glycosylation uses monosaccharides from all the above sources to produce nucleotide sugars required to assemble hundreds of distinct glycans. Here, we demonstrate that cells identify the origin/heritage of the monosaccharide, fucose, for glycosylation. We measured the contribution of GDP-fucose from each of these sources for glycan synthesis and found that different fucosyltransferases, individual glycoproteins, and linkage-specific fucose residues identify and select different GDP-fucose pools dependent on their heritage. This supports the hypothesis that GDP-fucose exists in multiple, distinct pools, not as a single homogenous pool. The selection is tightly regulated since the overall pool size remains constant. We present novel perspectives on monosaccharide metabolism, which may have a general applicability.

A Dominant Heterozygous Mutation in COG4 Causes Saul-Wilson Syndrome, a Primordial Dwarfism, and Disrupts Zebrafish Development via Wnt Signaling.

Saul-Wilson syndrome (SWS) is a rare, skeletal dysplasia with progeroid appearance and primordial dwarfism. It is caused by a heterozygous, dominant variant (p.G516R) in COG4, a subunit of the conserved oligomeric Golgi (COG) complex involved in intracellular vesicular transport. Our previous work has shown the intracellular disturbances caused by this mutation; however, the pathological mechanism of SWS needs further investigation. We sought to understand the molecular mechanism of specific aspects of the SWS phenotype by analyzing SWS-derived fibroblasts and zebrafish embryos expressing this dominant variant. SWS fibroblasts accumulate glypicans, a group of heparan sulfate proteoglycans (HSPGs) critical for growth and bone development through multiple signaling pathways. Consistently, we find that glypicans are increased in zebrafish embryos expressing the COG4 p.G516R variant. These animals show phenotypes consistent with convergent extension (CE) defects during gastrulation, shortened body length, and malformed jaw cartilage chondrocyte intercalation at larval stages. Since non-canonical Wnt signaling was shown in zebrafish to be related to the regulation of these processes by glypican 4, we assessed wnt levels and found a selective increase of wnt4 transcripts in the presence of COG4 p.G516R . Moreover, overexpression of wnt4 mRNA phenocopies these developmental defects. LGK974, an inhibitor of Wnt signaling, corrects the shortened body length at low concentrations but amplifies it at slightly higher concentrations. WNT4 and the non-canonical Wnt signaling component phospho-JNK are also elevated in cultured SWS-derived fibroblasts. Similar results from SWS cell lines and zebrafish point to altered non-canonical Wnt signaling as one possible mechanism underlying SWS pathology.

Protease-dependent defects in N-cadherin processing drive PMM2-CDG pathogenesis.

The genetic bases for the congenital disorders of glycosylation (CDG) continue to expand, but how glycosylation defects cause patient phenotypes remains largely unknown. Here, we combined developmental phenotyping and biochemical studies in a potentially new zebrafish model (pmm2sa10150) of PMM2-CDG to uncover a protease-mediated pathogenic mechanism relevant to craniofacial and motility phenotypes in mutant embryos. Mutant embryos had reduced phosphomannomutase activity and modest decreases in N-glycan occupancy as detected by matrix-assisted laser desorption ionization mass spectrometry imaging. Cellular analyses of cartilage defects in pmm2sa10150 embryos revealed a block in chondrogenesis that was associated with defective proteolytic processing, but seemingly normal N-glycosylation, of the cell adhesion molecule N-cadherin. The activities of the proconvertases and matrix metalloproteinases responsible for N-cadherin maturation were significantly altered in pmm2sa10150 mutant embryos. Importantly, pharmacologic and genetic manipulation of proconvertase activity restored matrix metalloproteinase activity, N-cadherin processing, and cartilage pathology in pmm2sa10150 embryos. Collectively, these studies demonstrate in CDG that targeted alterations in protease activity create a pathogenic cascade that affects the maturation of cell adhesion proteins critical for tissue development.

Cloning and characterization of an rRNA methyltransferase from Sorangium cellulosum.

A locus (kmr) responsible for aminoglycosides-resistance of Sorangium cellulosum was cloned and characterized in Myxococcus xanthus. The gene kmr encodes a putative rRNA methyltransferase. Expression of the complete ORF endowed the Myxococcus transformants with the resistance to aminoglycosidic antibiotics of kanamycin, apramycin, gentamycin, neomycin, and tobramycin at an extraordinary high-level (MIC, higher than 500 microg/ml). However, the gene did not function in Escherichia coli cells. In Sorangium genome, the gene kmr was followed by a putative integrase gene, and was highly homologous in different Sorangium strains. The Sorangium rRNA methyltransferase sequence was in low similarity to the reported 16S rRNA methyltransferases, and their resistance spectrums were also different. The results indicate that the rRNA methyltransferase (Kmr) in Sorangium strains is a new member of the rRNA methyltransferases family.

Discovery of the autonomously replicating plasmid pMF1 from Myxococcus fulvus and development of a gene cloning system in Myxococcus xanthus.

Myxobacteria are very important due to their unique characteristics, such as multicellular social behavior and the production of diverse and novel bioactive secondary metabolites. However, the lack of autonomously replicating plasmids has hindered genetic manipulation of myxobacteria for decades. To determine whether indigenous plasmids are present, we screened about 150 myxobacterial strains, and a circular plasmid designated pMF1 was isolated from Myxococcus fulvus 124B02. Sequence analysis showed that this plasmid was 18,634 bp long and had a G+C content of 68.7%. Twenty-three open reading frames were found in the plasmid, and 14 of them were not homologous to any known sequence. Plasmids containing the gene designated pMF1.14, which encodes a large unknown protein, were shown to transform Myxococcus xanthus DZ1 and DK1622 at high frequencies ( approximately 10(5) CFU/microg DNA), suggesting that the locus is responsible for the autonomous replication of pMF1. Shuttle vectors were constructed for both M. xanthus and Escherichia coli. The pilA gene, which is essential for pilus formation and social motility in M. xanthus, was cloned into the shuttle vectors and introduced into the pilA-deficient mutant DK10410. The transformants subsequently exhibited the ability to form pili and social motility. Autonomously replicating plasmid pMF1 provides a new tool for genetic manipulation in Myxococcus.

Evolutionary diversity of ketoacyl synthases in cellulolytic myxobacterium Sorangium.

The diversity of type I polyketide synthases (PKSs) in cellulolytic myxobacterium Sorangium was explored by assaying the ketoacyl synthases (KSs) in 10 Sorangium strains with two degenerate primer sets and 64 different KS fragments were obtained. For their deduced amino acid sequences, eight were identical to three known KSs from Sorangium and Magnetospirillum, while the others showed 54-83% identities to the modular KS domains reported from various microorganisms. Parts of the Sorangium KSs tightly share the clade with Actinobacteria excluding any other analyzed myxobacterial KSs, or with Cyanobacteria /Myxobacteria. Parts are widely located in the three functional groups - "Loading", "NRPS/PKS" and "Trans-AT". Sorangium KSs in the Actinobacteria, Cyanobacteria/Myxobacteria, or "Loading" clade further evolved independently on its own genus. Notably, the modular KSs from other Myxobacteria genera, i.e. Myxococcus, Stigmatella, Melittangium, Cystobacter and Angiococcus are often distributed crosswise and form non-Sorangium blend subgroups. "NRPS/PKS" and "Trans-AT" are two rather diverse groups and the Sorangium KSs in these clades evolved crosswise with other taxa lineages. The results presented in this paper suggest that the inherent genetic strategies, together with frequent gene importing from many organisms (HGT) have contributed to the evolution of modular PKSs in Sorangium. These findings reinforce that Sorangium strains are really excellent creators for novel and diverse polyketides.

TRIM37, a novel E3 ligase for PEX5-mediated peroxisomal matrix protein import.

Most proteins destined for the peroxisomal matrix depend on the peroxisomal targeting signals (PTSs), which require the PTS receptor PEX5, whose deficiency causes fatal human peroxisomal biogenesis disorders (PBDs). TRIM37 gene mutations cause muscle-liver-brain-eye (mulibrey) nanism. We found that TRIM37 localizes in peroxisomal membranes and ubiquitylates PEX5 at K464 by interacting with its C-terminal 51 amino acids (CT51), which is required for PTS protein import. PEX5 mutations (K464A or ΔCT51), or TRIM37 depletion or mutation, reduce PEX5 abundance by promoting its proteasomal degradation, thereby impairing its functions in cargo binding and PTS protein import in human cells. TRIM37 or PEX5 depletion induces apoptosis and enhances sensitivity to oxidative stress, underscoring the cellular requirement for functional peroxisomes. Therefore, TRIM37-mediated ubiquitylation stabilizes PEX5 and promotes peroxisomal matrix protein import, suggesting that mulibrey nanism is a new PBD.

Distinct requirements for intra-ER sorting and budding of peroxisomal membrane proteins from the ER.

During de novo peroxisome biogenesis, importomer complex proteins sort via two preperoxisomal vesicles (ppVs). However, the sorting mechanisms segregating peroxisomal membrane proteins to the preperoxisomal endoplasmic reticulum (pER) and into ppVs are unknown. We report novel roles for Pex3 and Pex19 in intra-endoplasmic reticulum (ER) sorting and budding of the RING-domain peroxins (Pex2, Pex10, and Pex12). Pex19 bridged the interaction at the ER between Pex3 and RING-domain proteins, resulting in a ternary complex that was critical for the intra-ER sorting and subsequent budding of the RING-domain peroxins. Although the docking subcomplex proteins (Pex13, Pex14, and Pex17) also required Pex19 for budding from the ER, they sorted to the pER independently of Pex3 and Pex19 and were spatially segregated from the RING-domain proteins. We also discovered a unique role for Pex3 in sorting Pex10 and Pex12, but with the docking subcomplex. Our study describes an intra-ER sorting process that regulates segregation, packaging, and budding of peroxisomal importomer subcomplexes, thereby preventing their premature assembly at the ER.