Eupatilin improves cilia defects in human CEP290 ciliopathy models.

The photoreceptor outer segment is a highly specialized primary cilium essential for phototransduction and vision. Biallelic pathogenic variants in the cilia-associated gene CEP290 cause non-syndromic Leber congenital amaurosis 10 (LCA10) and syndromic diseases, where the retina is also affected. While RNA antisense oligonucleotides and gene editing are potential treatment options for the common deep intronic variant c.2991+1655A>G in CEP290 , there is a need for variant-independent approaches that could be applied to a broader spectrum of ciliopathies. Here, we generated several distinct human models of CEP290 -related retinal disease and investigated the effects of the flavonoid eupatilin as a potential treatment. Eupatilin improved cilium formation and length in CEP290 LCA10 patient-derived fibroblasts, in gene-edited CEP290 knockout (CEP290 KO) RPE1 cells, and in both CEP290 LCA10 and CEP290 KO iPSCs-derived retinal organoids. Furthermore, eupatilin reduced rhodopsin retention in the outer nuclear layer of CEP290 LCA10 retinal organoids. Eupatilin altered gene transcription in retinal organoids, by modulating the expression of rhodopsin, and by targeting cilia and synaptic plasticity pathways. This work sheds light into the mechanism of action of eupatilin, and supports its potential as a variant-independent approach for CEP290 -associated ciliopathies.

Angiotensin mediates renal fibrosis in the nephropathy of glycogen storage disease type Ia.

Patients with glycogen storage disease type Ia (GSD-Ia) develop renal disease of unknown etiology despite intensive dietary therapies. This renal disease shares many clinical and pathological similarities to diabetic nephropathy. We studied the expression of angiotensinogen, angiotensin type 1 receptor, transforming growth factor-beta1, and connective tissue growth factor in mice with GSD-Ia and found them to be elevated compared to controls. While increased renal expression of angiotensinogen was evident in 2-week-old mice with GSD-Ia, the renal expression of transforming growth factor-beta and connective tissue growth factor did not increase for another week; consistent with upregulation of these factors by angiotensin II. The expression of fibronectin and collagens I, III, and IV was also elevated in the kidneys of mice with GSD-Ia, compared to controls. Renal fibrosis was characterized by a marked increase in the synthesis and deposition of extracellular matrix proteins in the renal cortex and histological abnormalities including tubular basement membrane thickening, tubular atrophy, tubular dilation, and multifocal interstitial fibrosis. Our results suggest that activation of the angiotensin system has an important role in the pathophysiology of renal disease in patients with GSD-Ia.

Long-term correction of murine glycogen storage disease type Ia by recombinant adeno-associated virus-1-mediated gene transfer.

Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in glucose-6-phosphatase-alpha (G6Pase-alpha), a nine-transmembrane domain, endoplasmic reticulum-associated protein expressed primarily in the liver and kidney. Previously, we showed that infusion of an adeno-associated virus (AAV) serotype 2 vector carrying murine G6Pase-alpha (AAV2-G6Pase-alpha) into neonatal GSD-Ia mice failed to sustain their life beyond weaning. We now show that neonatal infusion of GSD-Ia mice with an AAV serotype 1-G6Pase-alpha (AAV1-G6Pase-alpha) or AAV serotype 8-G6Pase-alpha (AAV8-G6Pase-alpha) results in hepatic expression of the G6Pase-alpha transgene and markedly improves the survival of the mice. However, only AAV1-G6Pase-alpha can achieve significant renal transgene expression. A more effective strategy, in which a neonatal AAV1-G6Pase-alpha infusion is followed by a second infusion at age one week, provides sustained expression of a complete, functional, G6Pase-alpha system in both the liver and kidney and corrects the metabolic abnormalities in GSD-Ia mice for the 57 week length of the study. This effective use of gene therapy to correct metabolic imbalances and disease progression in GSD-Ia mice holds promise for the future of gene therapy in humans.

In islet-specific glucose-6-phosphatase-related protein, the beta cell antigenic sequence that is targeted in diabetes is not responsible for the loss of phosphohydrolase activity.

AIMS/HYPOTHESIS: There are three members of the glucose-6-phosphatase (G6Pase) family: (1) the liver/kidney/intestine G6Pase-alpha (encoded by G6PC), which is a key enzyme in glucose homeostasis; (2) the ubiquitous G6Pase-beta (encoded by G6PC3); and (3) the islet-specific G6Pase-related protein (IGRP, encoded by /G6PC2). While G6Pase-alpha and G6Pase-beta are functional glucose-6-phosphate hydrolases, IGRP possesses almost no hydrolase activity. This was unexpected since G6Pase-alpha is more closely related to IGRP than G6Pase-beta. Recently, amino acids 206-214 in IGRP were identified as a beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes, suggesting that this peptide confers functional specificity to IGRP. We therefore investigated the molecular events that inactivate IGRP activity and the effects of the beta cell antigen sequence on the stability and enzymatic activity of G6Pase-alpha. METHODS: Studies were performed using site-directed mutagenesis and transient expression assays. Protein stability was evaluated by Western blotting, proteasome inhibitor studies and in vitro transcription-translation. RESULTS: We showed that the residues responsible for G6Pase activity are more extensive than previously recognised. Introducing the IGRP antigenic motif into G6Pase-alpha does not completely destroy activity, although it does destabilise the protein. The low hydrolytic activity in IGRP is due to the combination of multiple independent mutations. CONCLUSIONS/INTERPRETATION: The loss of catalytic activity in IGRP arises from the sum of many sequence differences. G6Pase-alpha mutants containing the beta cell antigen sequence are preferentially degraded in cells, which prevents targeting by pathogenic CD8+ T cells. It is possible that IGRP levels in beta cells could dictate susceptibilities to diabetes.

Pregnancy-specific glycoprotein gene expression in recurrent aborters: a potential correlation to interleukin-10 expression.

PROBLEM: The expression of the pregnancy-specific glycoprotein (PSG) genes in the uterine endometrium of women experiencing recurrent first-trimester abortions, and potential correlations to cytokine expression were examined. METHOD OF STUDY: Endometrial RNA, isolated from women with a history of either repetitive first-trimester pregnancy losses or uncomplicated pregnancies, was isolated and analyzed for PSG transcripts by the reverse transcriptase-polymerase chain reaction method. PSG genes showing different patterns of expression were expressed in baculovirus, and the purified proteins examined for their effects on cytokine expression. RESULTS: The expression of PSG11 in the endometria of recurrent aborters was significantly lower than in that of controls (P < 0.01). When tested on monocytes, PSG11 stimulated secretion of interleukin (IL)-10. CONCLUSIONS: The level of expression of the PSG11 gene in the uterine endometrium, during the peri-implantation period, correlates with the risk of pregnancy loss in some women experiencing recurrent spontaneous abortions. The ability of PSG11 to influence the secretion of IL-10 suggests that PSG11 may contribute to the local modulation of the inflammatory T helper-1 response seen in the endometrium of these women.

The gene for glycogen-storage disease type 1b maps to chromosome 11q23.

Glycogen-storage disease type 1 (GSD-1), also known as "von Gierke disease," is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase) activity. There are four distinct subgroups of this autosomal recessive disorder: 1a, 1b, 1c, and 1d. All share the same clinical manifestations, which are caused by abnormalities in the metabolism of glucose-6-phosphate (G6P). However, only GSD-1b patients suffer infectious complications, which are due to both the heritable neutropenia and the functional deficiencies of neutrophils and monocytes. Whereas G6Pase deficiency in GSD-1a patients arises from mutations in the G6Pase gene, this gene is normal in GSD-1b patients, indicating a separate locus for the disorder in the 1b subgroup. We now report the linkage of the GSD-1b locus to genetic markers spanning a 3-cM region on chromosome 11q23. Eventual molecular characterization of this disease will provide new insights into the genetic bases of G6P metabolism and neutrophil-monocyte dysfunction.

Glucose-6-phosphatase dependent substrate transport in the glycogen storage disease type-1a mouse.

Glycogen storage disease type 1a (GSD-1a) is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. A G6Pase knockout mouse which mimics the pathophysiology of human GSD-1a patients was created to understand the pathogenesis of this disorder, to delineate the mechanisms of G6Pase catalysis, and to develop future therapeutic approaches. By examining G6Pase in the liver and kidney, the primary gluconeogenic tissues, we demonstrate that glucose-6-P transport and hydrolysis are performed by separate proteins which are tightly coupled. We propose a modified translocase catalytic unit model for G6Pase catalysis.

Evolutionary analysis of the multigene pregnancy-specific beta 1-glycoprotein family: separation of historical and nonhistorical signals.

The pregnancy-specific beta 1-glycoproteins (PSG) form a large family of closely related proteins. Using newly developed methods of sequence analysis, in combination with protein modeling, we provide a framework for investigating the evolution and biological function of genes like the PSG. Evolutionary trees, based on C-terminal sequence, group PSG genes in a manner consistent with their genomic organization. Trees constructed using the N-terminal domain sequences are unreliable as an indicator of phylogeny because of non-neutral processes of sequence change. During duplication of the PSG genes, evolutionary pressures have resulted in a gradient of constrained change across each gene. The N-terminal domains show a nonrandom pattern of amino acid substitutions clustered in the immunoglobulin complementarity-determining region (CDR)-like regions, which appear to be important in the function of the protein.

A motif in PSG11s mediates binding to a receptor on the surface of the promonocyte cell line THP-1.

The pregnancy-specific glycoproteins (PSG) form a large family of essential pregnancy proteins, but their biological function is unknown. We have investigated whether one function of the PSG is to interact with cells of the maternal immune system. Normal human peripheral blood mononuclear cells, activated with phorbol ester, are shown to bind to purified placental PSG. This binding activity can be mimicked using a chemically synthesized peptide ligand containing the Arg-Gly-Asp (RGD) motif present in the N-terminal domain of PSG11s. The PSG11s receptors are present on cells of the myeloid cell lineage but not of the T cell or B cell lineages. The binding is mediated in part by the RGD motif and can be competed against by appropriate RGD-containing, but not Arg-Ala-Asp (RAD)-containing, ligands. Ligand binding requires a functional cytoskeleton. By examining the U937 and THP-1 promonocyte cell lines, the presence of receptors with two different binding characteristics are demonstrated. The THP-1 receptor is identified by chemical cross-linking as a protein of 46 kilodaltons (kDa), and affinity chromatography demonstrates the presence of three protein species of 32 kDa, 16.8 kDa, and 15.9 kDa, suggesting the receptor has multiple subunits.

Sequence of a novel pregnancy-specific beta 1-glycoprotein C-terminal domain.

A sequence related to the C-terminal coding regions of a subgroup 1 pregnancy-specific beta 1-glycoprotein (PSG) has been characterised upstream of the PSG11 gene. The sequence can encode the Cc, Ca, and Cb, domains but there appear to be no other PSG gene exons within at least 10 kb. Based on the organisation of other PGS genes, the position of this sequence is novel.

Characterization of the PSG11 gene.

The pregnancy-specific beta 1-glycoproteins (PSG) form the major group of proteins synthesized in the human placenta. There are over 30 proteins in the family, encoded by 11 genes located on chromosomes 19q13.1-13.3. The genes can be divided into three subgroups based on the C-terminal exons expressed. The subgroup 1 genes have been well characterized. In this study the organization and sequence of a complete, functional, subgroup 3 gene is described. It contains the C-terminal exons, Cw, Cr, and Cs, which are expected from the transcripts characterized. Down-stream from these exons are sequences homologous to the C-termini of the subgroup 1 type genes. This demonstrates that the subgroup 1, 2, and 3 genes are related via insertions/deletions. Comparison of the C-terminal sequences of the three subgroups of genes shows that the subgroup 2 and 3 genes are more closely related than, and are distinct from, the subgroup 1 genes.

The carboxyl-terminal domain of the human pregnancy-specific glycoprotein specifies intracellular retention and stability.

The pregnancy-specific glycoproteins (PSGs), which are members of the immunoglobulin superfamily, are the major pregnancy-associated proteins synthesized by the human placenta. Thirty or more PSG members have been identified which are encoded by at least 11 linked genes. The PSG proteins share 85-95% sequence homology in the coding region, but show variability at the carboxyl-terminal (COOH) domains. In the present study, we examined the effects of PSG COOH domains on protein secretion and stability. Using PSGs containing short (11-12 residues) hydrophilic (PSG1e, PSG11s, and PSG16a), short (22 residues) hydrophobic (PSG6r), and long (81 residues) hydrophobic (PSG11w) COOH domains, we showed that most PSG members were secretory proteins except PSG11w which was largely retained in cells. When the PSG11w COOH domain was replaced with a short COOH domain of PSG1e, the resulting PSG-N11w/C1e chimera became secreted into the medium. On the other hand, chimeras that harbored the PSG11w COOH domain, PSG-N1e/C11w and PSG-N16a/C11w, remained in cells, demonstrating that the COOH domain of PSG11w confers intracellular retention. Deletion analysis showed that mutant (PSG11w-C2) that contained the first 21 amino acids of PSG11w COOH domain or mutant (PSG11w-C3) that contained a deletion of hydrophobic residues 372-392 in the PSG11w COOH domain remained largely in cells. In contrast, the PSG11w-C1 mutant which contained the first 12 residues of the PSG11w COOH domain became a secretory protein. Studies of PSG synthesis and processing in the presence of Brefeldin A, a drug that impedes protein transport from endoplasmic reticulum to the Golgi system, showed that PSG11w resided and degraded in the endoplasmic reticulum. The endoplasmic reticulum localization of PSG11w and the cell-associated mutant PSGs was further demonstrated by their sensitivity to endoglycosidase H and indirect immunofluorescence analysis.

cDNA sequence of the pregnancy-specific beta 1-glycoprotein-11s (PSG-11s).

Four cDNA clones representing the human pregnancy-specific beta 1-glycoprotein-11 (PSG-11) gene have been characterised. All encoded a splice variant of the PSG-11 gene designated PSG-11s, which can encode a secreted protein of 426 amino acids, containing six potential N-linked glycosylation sites, with a domain structure L-N-AI-AII-BII-C. Minor differences between the four clones sequenced included a restriction site polymorphic for ApaI that may differentiate between alleles of the PSG-11 gene.

Isolation of intact larval haemoglobin from the brine shrimp Artemia salina. Prevention of degradation in vitro by proteases induced during larval development.

Haemoglobin induced in the larval stage of the brine shrimp, Artemia salina is extensively degraded when isolated from the later developmental stages of the larvae. Alkaline proteases appear in the organism a few hours after the induction of haemoglobin and cause the observed degradation. Addition of 2.6 mM phenylmethylsulphonyl fluoride or 20 micrograms/ml soybean trypsin inhibitor to the extraction buffer used for haemoglobin isolation prevents most of this degradation. Discrete haem proteins are found in extracts of the brine shrimp larvae isolated before induction of the proteases, and the major species has a molecular weight of over 200,000. This is believed to be the native haemoglobin. A spread of lower molecular weight haem-containing polypeptides is found in extracts of larvae isolated after induction of the proteases. These products are believed to result from degradation of the discrete haem proteins present in protease-free extracts.