The cation-independent mannose-6-phosphate/IGF2 receptor (CI-M6P/IGF2R) plays a crucial role in transporting lysosomal enzymes and other ligands. In this study, we designed and synthesized novel stable mannose-6-phosphate (M6P) derivatives to enhance their affinity for CI-M6P/IGF2R. To evaluate the binding potency, we employed a sensitive and cost-effective fluorescence polarization assay, enabling rapid quantification of receptor-ligand interactions in solution. The tested compounds included di-, tri-, and penta-M6P peptides along with various M6P-derived small molecules featuring phosphate isosteres or other functional modifications. Our findings indicate that ligands bearing multiple M6P moieties exhibit significantly higher receptor affinities than monomeric compounds and that phosphonate groups may serve as a more stable and potent alternative to native M6P. Computational modeling of ligand interactions with the CI-M6P/IGF2R domains further elucidated the binding mechanisms, offering new directions for the development of more effective ligands. This study advances the design of therapeutic strategies that leverage CI-M6P/IGF2R for targeted biomolecule delivery to lysosomes, thereby opening new possibilities for biomedical applications.

• Klíčová slova
• IGF2, Mannose‐6‐phosphate, fluorescence polarization assay, ligand binding, receptor,
• MeSH
• fluorescenční polarizace MeSH
• lidé MeSH
• ligandy MeSH
• lyzozomy metabolismus   MeSH
• mannosafosfáty * chemie   metabolismus   chemická syntéza   MeSH
• racionální návrh léčiv MeSH
• receptor IGF typ 2 * metabolismus   chemie   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• IGF2R protein, human MeSH Prohlížeč
• ligandy MeSH
• mannosafosfáty * MeSH
• mannose-6-phosphate MeSH Prohlížeč
• receptor IGF typ 2 * MeSH

It was shown that hexokinase contained inside permeabilized yeast cells can be used successfully in the phosphorylation of mannose for the production of mannose 6-phosphate. We propose that enzymes entrapped within the semipermeable cell membrane be considered as an extension of the enzyme immobilization concept, since it shares many of its advantages and several of its properties with some unique characteristics.

• MeSH
• buněčná membrána enzymologie   MeSH
• dimethylsulfoxid farmakologie   MeSH
• enzymy imobilizované metabolismus   MeSH
• fosforylace MeSH
• hexokinasa metabolismus   MeSH
• hexosafosfáty biosyntéza   MeSH
• mannosa-6-fosfátisomerasa genetika   MeSH
• mannosa metabolismus   MeSH
• mannosafosfáty biosyntéza   MeSH
• mutace MeSH
• permeabilita buněčné membrány účinky léků   MeSH
• Saccharomyces cerevisiae účinky léků   enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dimethylsulfoxid MeSH
• enzymy imobilizované MeSH
• hexokinasa MeSH
• hexosafosfáty MeSH
• mannosa-6-fosfátisomerasa MeSH
• mannosa MeSH
• mannosafosfáty MeSH
• mannose-6-phosphate MeSH Prohlížeč

The delivery of newly synthesized soluble lysosomal hydrolases to the endosomal system is essential for lysosome function and cell homeostasis. This process relies on the proper trafficking of the mannose 6-phosphate receptors (MPRs) between the trans-Golgi network (TGN), endosomes and the plasma membrane. Many transmembrane proteins regulating diverse biological processes ranging from virus production to the development of multicellular organisms also use these pathways. To explore how cell signaling modulates MPR trafficking, we used high-throughput RNA interference (RNAi) to target the human kinome and phosphatome. Using high-content image analysis, we identified 127 kinases and phosphatases belonging to different signaling networks that regulate MPR trafficking and/or the dynamic states of the subcellular compartments encountered by the MPRs. Our analysis maps the MPR trafficking pathways based on enzymes regulating phosphatidylinositol phosphate metabolism. Furthermore, it reveals how cell signaling controls the biogenesis of post-Golgi tubular carriers destined to enter the endosomal system through a SRC-dependent pathway regulating ARF1 and RAC1 signaling and myosin II activity.

• Klíčová slova
• Lysosome, Mannose 6-phosphate receptor, Post-Golgi transport, Trafficking, siRNA screen,
• MeSH
• ADP-ribosylační faktor 1 genetika   metabolismus   MeSH
• buněčná membrána enzymologie   MeSH
• endozomy enzymologie   MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• genové regulační sítě MeSH
• HeLa buňky MeSH
• lidé MeSH
• mapy interakcí proteinů MeSH
• rac1 protein vázající GTP genetika   metabolismus   MeSH
• receptor IGF typ 2 genetika   metabolismus   MeSH
• regulace genové exprese enzymů MeSH
• RNA interference * MeSH
• shluková analýza MeSH
• signální transdukce MeSH
• skupina kinas odvozených od src-genu genetika   metabolismus   MeSH
• trans-Golgiho síť enzymologie   MeSH
• transfekce MeSH
• transport proteinů genetika   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ADP-ribosylační faktor 1 MeSH
• fosfatidylinositolfosfáty MeSH
• rac1 protein vázající GTP MeSH
• RAC1 protein, human MeSH Prohlížeč
• receptor IGF typ 2 MeSH
• skupina kinas odvozených od src-genu MeSH

The plasminogen system is harnessed in a wide variety of physiological processes, such as fibrinolysis, cell migration, or efferocytosis; and accordingly, it is essential upon inflammation, tissue remodeling, wound healing, and for homeostatic maintenance in general. Previously, we identified a plasminogen receptor in the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R, CD222). Here, we demonstrate by means of genetic knockdown, knockout, and rescue approaches combined with functional studies that M6P/IGF2R is up-regulated on the surface of macrophages, recognizes plasminogen exposed on the surface of apoptotic cells, and mediates plasminogen-induced efferocytosis. The level of uptake of plasminogen-coated apoptotic cells inversely correlates with the TNF-α production by phagocytes indicating tissue clearance without inflammation by this mechanism. Our results reveal an up-to-now undetermined function of M6P/IGF2R in clearance of apoptotic cells, which is crucial for tissue homeostasis.

• Klíčová slova
• M6P/IGF2R, efferocytosis, macrophages, plasminogen, tissue homeostasis,
• MeSH
• buněčná diferenciace účinky léků   MeSH
• fagocytóza účinky léků   MeSH
• fibroblasty účinky léků   metabolismus   MeSH
• genový knockout MeSH
• Jurkat buňky MeSH
• lidé MeSH
• makrofágy cytologie   účinky léků   metabolismus   MeSH
• myši MeSH
• plazminogen farmakologie   MeSH
• receptor IGF typ 2 metabolismus   MeSH
• THP-1 buňky MeSH
• TNF-alfa metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• plazminogen MeSH
• receptor IGF typ 2 MeSH
• TNF-alfa MeSH

A plant selection system based on the phosphomannose isomerase gene (pmi) as a selectable marker is often used to avoid selection using antibiotic resistance. Nevertheless, pmi gene is endogenous in several plant species and therefore difficult to use in such cases. Here we evaluated and compared Agrobacterium-mediated transformation of Linum usitatissimum breeding line AGT-952 (without endogenous pmi gene) and Nicotiana tabacum var. WSC-38 (with endogenous pmi gene). Transformation was evaluated for vectors bearing transgenes that have the potential to be involved in improved phytoremediation of contaminated environment. Tobacco regenerants selection resulted in 6.8% transformation efficiency when using a medium supplemented with 30 g/L mannose with stepwise decrease of the sucrose concentration. Similar transformation efficiency (5.3%) was achieved in transformation of flax. Relatively low selection efficiency was achieved (12.5% and 34.8%, respectively). The final detection of efficient pmi selection was conducted using PCR and the non-endogenous genes; pmi transgene for flax and todC2 transgene for tobacco plants.

• Klíčová slova
• Linum usitatissimum, nicotiana tabacum, phosphomannose isomerase, positive selection system,
• MeSH
• Agrobacterium genetika   MeSH
• bakteriální transformace genetika   MeSH
• biodegradace MeSH
• geneticky modifikované rostliny genetika   MeSH
• kultivační média chemie   MeSH
• len účinky léků   genetika   MeSH
• mannosa-6-fosfátisomerasa genetika   MeSH
• mannosa metabolismus   farmakologie   MeSH
• selekce (genetika) MeSH
• tabák účinky léků   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kultivační média MeSH
• mannosa-6-fosfátisomerasa MeSH
• mannosa MeSH

A hexokinase preparation was obtained from a Saccharomyces cerevisiae mutant strain deficient in glucosephosphate isomerase (GPI) and mannosephosphate isomerase (MPI) by precipitation with ammonium sulfate. The supernatant fraction corresponding to 40-60 % saturation showed the lowest content in GPI and MPI activity. The fraction was used without further purification in the determination of glucose, either free or in a mixture with fructose and mannose. The results were similar to those obtained with pure commercial hexokinase.

• MeSH
• chemická precipitace MeSH
• glukosa-6-fosfátisomerasa genetika   MeSH
• hexokinasa izolace a purifikace   MeSH
• mannosa-6-fosfátisomerasa genetika   MeSH
• mutace MeSH
• Saccharomyces cerevisiae enzymologie   genetika   MeSH
• síran amonný MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glukosa-6-fosfátisomerasa MeSH
• hexokinasa MeSH
• mannosa-6-fosfátisomerasa MeSH
• síran amonný MeSH

Mannose phosphate isomerase-congenital disorder of glycosylation (MPI-CDG) deficiency is a rare subtype of congenital disorders of protein N-glycosylation. It is characterised by deficiency of MPI caused by pathogenic variants in MPI gene. The manifestation of MPI-CDG is different from other CDGs as the patients suffer dominantly from gastrointestinal and hepatic involvement whereas they usually do not present intellectual disability or neurological impairment. It is also one of the few treatable subtypes of CDGs with proven effect of oral mannose. This article covers a complex review of the literature and recommendations for the management of MPI-CDG with an emphasis on the clinical aspect of the disease. A team of international experts elaborated summaries and recommendations for diagnostics, differential diagnosis, management, and treatment of each system/organ involvement based on evidence-based data and experts' opinions. Those guidelines also reveal more questions about MPI-CDG which need to be further studied.

• Klíčová slova
• AT deficiency, MPI-CDG, guidelines, hepatic fibrosis, hyperinsulinaemic hypoglycaemia, mannose phosphate isomerase, protein-losing enteropathy,
• MeSH
• konsensus MeSH
• lidé MeSH
• management nemoci MeSH
• mannosa-6-fosfátisomerasa nedostatek   genetika   MeSH
• směrnice pro lékařskou praxi jako téma MeSH
• vrozené poruchy glykosylace diagnóza   enzymologie   terapie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• mannosa-6-fosfátisomerasa MeSH

A positive selection system using phosphomannose isomerase was employed for Agrobacterium tumefaciens mediated transformation of lettuce (Lactuca sativa L. var. 'Achát'). It was shown that the mannose-based selection system works very well with the lettuce genotype used, reaching up to 25% transformation efficiency on the medium with 20 g/L mannose and 20 g/L sucrose. The best transformation efficacy with the commonly-used kanamycin at 100 mg/L as a selection agent was 21%. Southern blot analyses of thirteen chosen mannose-resistant regenerants revealed that some of them have clonal origin, about one-half harbour a single T-DNA copy and one plant contains an incomplete T-DNA segment with only the left part of T-DNA with the pmi gene present in the genomic DNA. The following Northern analysis showed transcriptional activity of the introduced pmi gene in all plants analysed with very high differences in the level of pmi specific mRNA. The results demonstrate that both mannose and kanamycin provide comparable transformation efficiencies in our lettuce genotype. An alternative selection method with mannose as a selection agent is now available for lettuce transgenosis.

• MeSH
• Agrobacterium tumefaciens genetika   MeSH
• geneticky modifikované rostliny MeSH
• mannosa-6-fosfátisomerasa genetika   MeSH
• salát (hlávkový) enzymologie   genetika   růst a vývoj   MeSH
• selekce (genetika) * MeSH
• transformace genetická * MeSH
• výhonky rostlin enzymologie   genetika   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mannosa-6-fosfátisomerasa MeSH

The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases.

• Klíčová slova
• Trichomonas vaginalis, cysteine peptidase, glycosylation, mannose 6-phosphate receptor, phagolysosome, proteome,
• MeSH
• cystein metabolismus   MeSH
• cysteinové proteasy * metabolismus   MeSH
• fagozomy metabolismus   MeSH
• lidé MeSH
• lyzozomy metabolismus   MeSH
• proteasy metabolismus   MeSH
• proteomika MeSH
• Trichomonas vaginalis * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystein MeSH
• cysteinové proteasy * MeSH
• proteasy MeSH

Insulin-like growth factor 2 receptor (Igf2r), a maternally expressed imprinted gene, is triplicated in Ts43H trisomic mice. To verify its expression in a trisomic mode, we examined the allele-specific transcripts and relative levels of Igf2r mRNA in trisomic and control mice. Igf2r was expressed from the maternal allele(s) and silenced on the paternal allele(s) in most tissues of maternally or paternally derived Ts43H mice. The triallelic expression was observed in adult brain and testis and in embryonic head, but the antisense transcript, Air, was strictly paternal, and methylation of region 2 was strictly maternal in all tissues. The Igf2r expression in maternally derived trisomics, with two active copies of the gene, did not exceed the average mRNA levels of euploid controls with monoallelic expression. Thus, an indication is presented for a dosage compensation mechanism of Igf2r in the trisomic context.

• MeSH
• antisense RNA metabolismus   MeSH
• genomový imprinting * MeSH
• metylace DNA * MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• orgánová specificita MeSH
• receptor IGF typ 2 genetika   metabolismus   MeSH
• trizomie genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antisense RNA MeSH
• receptor IGF typ 2 MeSH