Production of Domain 9 from the cation-independent mannose-6-phosphate receptor fused with an Fc domain.

Lysosomal storage diseases (LSDs) are genetic disorders caused by mutations in lysosomal enzymes, lysosomal membrane proteins or genes related to intracellular transport that result in impaired lysosomal function. Currently, the primary treatment for several LSDs is enzyme replacement therapy (ERT), which involves intravenous administration of the deficient lysosomal enzymes to ameliorate symptoms. The efficacy of ERT largely depends on the mannose-6-phosphate (M6P) modification of the N-glycans associated with the enzyme, as M6P is a marker for the recognition and trafficking of lysosomal enzymes. In cells, N-glycan processing and M6P modification occur in the endoplasmic reticulum and Golgi apparatus. This is a complex process involving multiple enzymes. In the trans-Golgi network (TGN), M6P-modified enzymes are recognized by the cation-independent mannose-6-phosphate receptor (CIMPR) and transported to the lysosome to exert their activities. In this study, we used the 9th domain of CIMPR, which exhibits a high affinity for M6P binding, and fused it with the Fc domain of human immunoglobulin G1 (IgG1). The resulting fusion protein specifically binds to M6P-modified proteins. This provides a tool for the rapid detection and concentration of M6P-containing recombinant enzymes to assess the effectiveness of ERT. The advantages of this approach include its high specificity and sensitivity and may lead to the development of new treatments for LSDs.

Semi-rational engineering of D-allulose 3-epimerase for simultaneously improving the catalytic activity and thermostability based on D-allulose biosensor.

BACKGROUND: D-Allulose is one of the most well-known rare sugars widely used in food, cosmetics, and pharmaceutical industries. The most popular method for D-allulose production is the conversion from D-fructose catalyzed by D-allulose 3-epimerase (DAEase). To address the general problem of low catalytic efficiency and poor thermostability of wild-type DAEase, D-allulose biosensor was adopted in this study to develop a convenient and efficient method for high-throughput screening of DAEase variants. RESULTS: The catalytic activity and thermostability of DAEase from Caballeronia insecticola were simultaneously improved by semi-rational molecular modification. Compared with the wild-type enzyme, DAEaseS37N/F157Y variant exhibited 14.7% improvement in the catalytic activity and the half-time value (t1/2) at 65°C increased from 1.60 to 27.56 h by 17.23-fold. To our delight, the conversion rate of D-allulose was 33.6% from 500-g L-1 D-fructose in 1 h by Bacillus subtilis WB800 whole cells expressing this DAEase variant. Furthermore, the practicability of cell immobilization was evaluated and more than 80% relative activity of the immobilized cells was maintained from the second to seventh cycle. CONCLUSION: All these results indicated that the DAEaseS37N/F157Y variant would be a potential candidate for the industrial production of D-allulose.

Rft1 catalyzes lipid-linked oligosaccharide translocation across the ER membrane.

The eukaryotic asparagine (N)-linked glycan is pre-assembled as a fourteen-sugar oligosaccharide on a lipid carrier in the endoplasmic reticulum (ER). Seven sugars are first added to dolichol pyrophosphate (PP-Dol) on the cytoplasmic face of the ER, generating Man5GlcNAc2-PP-Dol (M5GN2-PP-Dol). M5GN2-PP-Dol is then flipped across the bilayer into the lumen by an ER translocator. Genetic studies identified Rft1 as the M5GN2-PP-Dol flippase in vivo but are at odds with biochemical data suggesting Rft1 is dispensable for flipping in vitro. Thus, the question of whether Rft1 plays a direct or an indirect role during M5GN2-PP-Dol translocation has been controversial for over two decades. We describe a completely reconstituted in vitro assay for M5GN2-PP-Dol translocation and demonstrate that purified Rft1 catalyzes the translocation of M5GN2-PP-Dol across the lipid bilayer. These data, combined with in vitro results demonstrating substrate selectivity and rft1∆ phenotypes, confirm the molecular identity of Rft1 as the M5GN2-PP-Dol ER flippase.

Production of CA125 with Tn antigens using a glycosylphosphatidylinositol anchoring system.

Cancer antigen 125 (CA125) is a serum marker associated with ovarian cancer. Despite its widespread use, CA125 levels can also be elevated in benign conditions. Recent reports suggest that detecting serum CA125 that carries the Tn antigen, a truncated O-glycan containing only N-acetylgalactosamine on serine or threonine residues, can improve the specificity of ovarian cancer diagnosis. In this study, we engineered cells to express CA125 with a Tn antigen. To achieve this, we knocked out C1GALT1 and SLC35A1, genes encoding Core1 synthase and a transporter for cytidine-5'-monophospho-sialic acid respectively, in human embryonic kidney 293 (HEK293) cells. In ClGALT1-SLC35A1-knockout (KO) cells, the expression of the Tn antigen showed a significant increase, whereas the expression of the T antigen (galactose-ß1,3-N-acetylgalactosamine on serine or threonine residues) was decreased. Due to the inefficient secretion of soluble CA125, we employed a glycosylphosphatidylinositol (GPI) anchoring system. This allowed for the expression of GPI-anchored CA125 on the cell surface of ClGALT1-SLC35A1-KO cells. Cells expressing high levels of GPI-anchored CA125 were then enriched through cell sorting. By knocking out the PGAP2 gene, the GPI-anchored form of CA125 was converted to a secretory form. Through the engineering of O-glycans and the use of a GPI-anchoring system, we successfully produced CA125 with Tn antigen modification.

Golgi α-mannosidases regulate cell surface N-glycan type and ectodomain shedding of the transmembrane protease corin.

Corin is a transmembrane protease that activates natriuretic peptides on the cell membrane. Reduced cell surface targeting or increased ectodomain shedding disrupts cell membrane homeostasis of corin, thereby impairing its cell surface expression and enzyme activity. N-glycans are essential in corin ectodomain shedding. Lack of N-glycans promotes corin ectodomain shedding in the juxtamembrane and frizzled-1 domains. The nascent N-glycans, transferred onto the polypeptide of corin, undergo multistep N-glycan processing in the endoplasmic reticulum and Golgi. It remains unclear how trimming by Golgi α-mannosidases, the critical N-glycan processing steps in N-glycan maturation, may regulate corin biosynthesis. In this study, we examined the effects of kifunensine and swainsonine, the inhibitors for α-mannosidases I and II, on corin expression and function. Western analysis of corin proteins in cell lysates and conditioned media from the inhibitor-treated corin-stable HEK293 cells and AC16 cells showed that both α-mannosidases I and II were required to maintain complex N-glycans on cell surface corin and protect corin from ectodomain shedding in the juxtamembrane and frizzled-1 domains. Cell viability analysis revealed that inhibition of α-mannosidase I or II sensitized cardiomyocytes to hydrogen peroxide-induced injury via regulating corin. Moreover, either one of the two coding genes was sufficient to perform Golgi α-mannosidase I trimming of N-glycans on corin. Similarly, this sufficiency was observed in Golgi α-mannosidase II-coding genes. Inhibition of ectodomain shedding restored corin zymogen activation from kifunensine- or swainsonine-induced reduction. Together, our results show the important roles of Golgi α-mannosidases in maintaining cell membrane homeostasis and biological activities of corin.

Comprehensive in silico analysis of glycosylphosphatidylinositol- anchored protein (GPI-AP) related genes expression profiles in human normal and cancer tissues.

In human cells, there are more than 146 glycosylphosphatidylinositol-anchored proteins (GPI-APs), including receptors, ligands, adhesion molecules and enzymes. The proteins are associated with membrane microdomains called lipid rafts through GPI, and plays a variety of important biological functions. At present, plenty of studies have been carried out on the biosynthesis of GPI-APs. The biosynthesis of GPI-APs requires at least 20 steps, and more than 40 GPI biosynthetic genes have been identified. However, it remains unclear how expression of GPI-AP related genes is regulated in normal and cancer tissues. In this study, we utilized gene expression data from both the TCGA database and GTEx portal to analysis the gene expression involved in GPI-AP biosynthesis and encoding GPI-APs in normal and cancer tissues. In order to perform a comprehensive analysis, we employed the GlycoMaple, a tool that is specifically designed to analyze glycosylation pathways. The results showed that compared with normal tissues, the expression of genes involved in GPI-AP biosynthesis in cancer tissues such as early glioma, glioblastoma multiforme, pancreatic cancer, testicular germ cell carcinoma, skin primary cutaneous melanoma and skin metastatic cutaneous melanoma, was changed significantly. Particularly, the expression of PIGY in these six cancers was increased. In addition, the expression of CD14, a GPI-AP gene, was increased in these six cancers. The expression of GAS1, GPC2 and GPC4 was increased only in early glioma and glioblastoma multiforme indicating that some GPI-APs such as GAS1 can be used as biomarkers of glioma. This study provides new insights into the expression of GPI-AP related genes in normal and cancer tissues, and lays a solid foundation for the development of GPI-APs as biomarkers.

A lipid scramblase TMEM41B is involved in the processing and transport of GPI-anchored proteins.

Protein modification by glycosylphosphatidylinositol (GPI) takes place in the endoplasmic reticulum (ER). GPI-anchored proteins (GPI-APs) formed in the ER are transported to the cell surface through the Golgi apparatus. During transport, the GPI-anchor structure is processed. In most cells, an acyl chain modified to the inositol of GPI is removed by a GPI-inositol deacylase, PGAP1, in the ER. Inositol-deacylated GPI-APs become sensitive to bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). We previously reported that GPI-APs are partially resistant to PI-PLC when PGAP1 activity is weakened by the deletion of selenoprotein T (SELT) or cleft lip and palate transmembrane protein 1 (CLPTM1). In this study, we found that the loss of TMEM41B, an ER-localized lipid scramblase, restored PI-PLC sensitivity of GPI-APs in SELT-knockout (KO) and CLPTM1-KO cells. In TMEM41B-KO cells, the transport of GPI-APs as well as transmembrane proteins from the ER to the Golgi was delayed. Furthermore, the turnover of PGAP1, which is mediated by ER-associated degradation, was slowed in TMEM41B-KO cells. Taken together, these findings indicate that inhibition of TMEM41B-dependent lipid scrambling promotes GPI-AP processing in the ER through PGAP1 stabilization and slowed protein trafficking.

Construction of a Yeast Cell-Based Assay System to Analyze SNAP25-Targeting Botulinum Neurotoxins.

Herein, we describe a yeast cell-based assay system to analyze SNAP25-targeting botulinum neurotoxins (BoNTs). BoNTs are protein toxins, and, upon incorporation into neuronal cells, their light chains (BoNT-LCs) target specific synaptosomal N-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins, including synaptosomal-associated protein 25 (SNAP25). BoNT-LCs are metalloproteases, and each BoNT-LC recognizes and cleaves conserved domains in SNAREs termed the SNARE domain. In the budding yeast Saccharomyces cerevisiae, the SNAP25 ortholog Spo20 is required for production of the spore plasma membrane; thus, defects in Spo20 cause sporulation deficiencies. We found that chimeric SNAREs in which SNARE domains in Spo20 are replaced with those of SNAP25 are functional in yeast cells. The Spo20/SNAP25 chimeras, but not Spo20, are sensitive to digestion by BoNT-LCs. We demonstrate that spo20∆ yeasts harboring the chimeras exhibit sporulation defects when various SNAP25-targeting BoNT-LCs are expressed. Thus, the activities of BoNT-LCs can be assessed by colorimetric measurement of sporulation efficiencies. Although BoNTs are notorious toxins, they are also used as therapeutic and cosmetic agents. Our assay system will be useful for analyzing novel BoNTs and BoNT-like genes, as well as their manipulation.

Studies on the Proteinaceous Structure Present on the Surface of the Saccharomyces cerevisiae Spore Wall.

The surface of the Saccharomyces cerevisiae spore wall exhibits a ridged appearance. The outermost layer of the spore wall is believed to be a dityrosine layer, which is primarily composed of a crosslinked dipeptide bisformyl dityrosine. The dityrosine layer is impervious to protease digestion; indeed, most of bisformyl dityrosine molecules remain in the spore after protease treatment. However, we find that the ridged structure is removed by protease treatment. Thus, a ridged structure is distinct from the dityrosine layer. By proteomic analysis of the spore wall-bound proteins, we found that hydrophilin proteins, including Sip18, its paralog Gre1, and Hsp12, are present in the spore wall. Mutant spores with defective hydrophilin genes exhibit functional and morphological defects in their spore wall, indicating that hydrophilin proteins are required for the proper organization of the ridged and proteinaceous structure. Previously, we found that RNA fragments were attached to the spore wall in a manner dependent on spore wall-bound proteins. Thus, the ridged structure also accommodates RNA fragments. Spore wall-bound RNA molecules function to protect spores from environmental stresses.

Accumulated precursors of specific GPI-anchored proteins upregulate GPI biosynthesis with ARV1.

We previously reported that glycosylphosphatidylinositol (GPI) biosynthesis is upregulated when endoplasmic reticulum-associated degradation (ERAD) is defective; however, the underlying mechanistic basis remains unclear. Based on a genome-wide CRISPR-Cas9 screen, we show that a widely expressed GPI-anchored protein CD55 precursor and ER-resident ARV1 are involved in upregulation of GPI biosynthesis under ERAD-deficient conditions. In cells defective in GPI transamidase, GPI-anchored protein precursors fail to obtain GPI, with the remaining uncleaved GPI-attachment signal at the C-termini. We show that ERAD deficiency causes accumulation of the CD55 precursor, which in turn upregulates GPI biosynthesis, where the GPI-attachment signal peptide is the active element. Among the 31 GPI-anchored proteins tested, only the GPI-attachment signal peptides of CD55, CD48, and PLET1 enhance GPI biosynthesis. ARV1 is prerequisite for the GPI upregulation by CD55 precursor. Our data indicate that GPI biosynthesis is balanced to need by ARV1 and precursors of specific GPI-anchored proteins.

Distributed Fault-Tolerant Formation Tracking Control for Multiagent Systems With Multiple Leaders and Constrained Actuators.

The distributed formation tracking control problem with multiple leaders under actuator faults and constraints is investigated in this article. All followers in the multiagent system should achieve a desired time-varying formation and track the convex combination of multiple leaders. To accomplish the control task, an active reconfigurable control scheme is proposed using the local information between agents, as well as the fault values of individuals provided by fault estimation observers. Combining with the Lyapunov stability theorem and the property of the Laplacian matrix, the control gains are calculated using the adaptive technique with a formation tracking feasibility condition. The original reconfigurable protocol is modified by utilizing anti-windup compensators to against saturation phenomenons (both magnitude and rate) in actuators. The simulation results validate that the presented scheme can address the faults as well as the actuator saturation.

Improvement of angle random walk of fiber-optic gyroscope using polarization-maintaining fiber ring resonator.

For an interferometric fiber-optic gyroscope (IFOG), the angle random walk, which represents the sensitivity of rotation detection, is mainly limited by the relative intensity noise (RIN) of a broadband source. Using a single-mode fiber ring resonator (SM-FRR) to filter the spectrum of a broadband light source is a common strategy for reducing the RIN at the proper IFOG frequency. However, this method depends on the polarization cross-coupling within the SM-FRR. We model the effect of polarization cross-coupling on the SM-FRR. Then, to further reduce the RIN, we introduce a polarization-maintaining fiber ring resonator (PM-FRR), which mitigates the effect of polarization cross-coupling on the SM-FRR. Using the PM-FRR as a spectrum filter, the RIN is reduced to -143 dB/Hz, with a reduction ratio of 25 dB, and the angle random walk in the IFOG is improved by over five times from 1.17 to 0.223 mdeg/h1/2 using a 2.1 km sensing coil.

Genome-wide CRISPR screen reveals CLPTM1L as a lipid scramblase required for efficient glycosylphosphatidylinositol biosynthesis.

SignificanceScramblases translocate lipids across the lipid bilayer without consumption of ATP, thereby regulating lipid distributions in cellular membranes. Cytosol-to-lumen translocation across the endoplasmic reticulum (ER) membrane is a common process among lipid glycoconjugates involved in posttranslational protein modifications in eukaryotes. These translocations are thought to be mediated by specific ER-resident scramblases, but the identity of these proteins and the underlying molecular mechanisms have been elusive. Here, we show that CLPTM1L, an integral membrane protein with eight putative transmembrane domains, is the major lipid scramblase involved in efficient glycosylphosphatidylinositol biosynthesis in the ER membrane. Our results validate the long-standing hypothesis that lipid scramblases ensure the efficient translocations of lipid glycoconjugates across the ER membrane for protein glycosylation pathways.

Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit.

Glycosylphosphatidylinositol (GPI) anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes. The biosynthesis and transfer of GPI to proteins are carried out in the endoplasmic reticulum. Attachment of GPI to proteins is mediated by the GPI-transamidase (GPI-TA) complex, which recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins. Then, GPI is transferred to the newly exposed C-terminus of the proteins. GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity. Here, we analyzed functionally important residues of the five subunits of GPI-TA by comparing conserved sequences among homologous proteins. In addition, we optimized the purification method for analyzing the structure of GPI-TA. Using purified GPI-TA, preliminary single particle images were obtained. Our results provide guidance for the structural and functional analysis of GPI-TA.

Simultaneous Trapping of Two Types of Particles with Focused Elegant Third-Order Hermite-Gaussian Beams.

The focusing properties of elegant third-order Hermite-Gaussian beams (TH3GBs) and the radiation forces exerted on dielectric spherical particles produced by such beams in the Rayleigh scattering regime have been theoretically studied. Numerical results indicate that the elegant TH3GBs can be used to simultaneously trap and manipulate nanosized dielectric spheres with refractive indexes lower than the surrounding medium at the focus and those with refractive indexes larger than the surrounding medium in the focal vicinity. Furthermore, by changing the radius of the beam waist, the transverse trapping range and stiffness at the focal plane can be changed.

A knockout cell library of GPI biosynthetic genes for functional studies of GPI-anchored proteins.

Over 100 kinds of proteins are expressed as glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) on the cell surface in mammalian cells. GPI-APs possess unique properties in terms of their intracellular trafficking and association with lipid rafts. Although it is clear that GPI-APs play critical roles in various biological phenomena, it is poorly understood how the GPI moiety contributes to these mechanisms. More than 30 genes are involved in the correct biosynthesis of GPI-APs. We here constructed a cell library in which 32 genes involved in GPI biosynthesis were knocked out in human embryonic kidney 293 cells. Using the cell library, the surface expression and sensitivity to phosphatidylinositol-specific phospholipase C of GPI-APs were analyzed. Furthermore, we identified structural motifs of GPIs that are recognized by a GPI-binding toxin, aerolysin. The cell-based GPI-knockout library could be applied not only to basic researches, but also to applications and methodologies related to GPI-APs.

Human SND2 mediates ER targeting of GPI-anchored proteins with low hydrophobic GPI attachment signals.

Over 100 glycosylphosphatidylinositol-anchored proteins (GPI-APs) are encoded in the mammalian genome. It is not well understood how these proteins are targeted and translocated to the endoplasmic reticulum (ER). Here, we reveal that many GPI-APs, such as CD59, CD55, and CD109, utilize human SND2 (hSND2)-dependent ER targeting machinery. We also found that signal recognition particle receptors seem to cooperate with hSND2 to target GPI-APs to the ER. Both the N-terminal signal sequence and C-terminal GPI attachment signal of GPI-APs contribute to ER targeting via the hSND2-dependent pathway. Particularly, the hydrophobicity of the C-terminal GPI attachment signal acts as the determinant of hSND2 dependency. Our results explain the route and mechanism of the ER targeting of GPI-APs in mammalian cells.

Global mapping of glycosylation pathways in human-derived cells.

Glycans are one of the fundamental classes of macromolecules and are involved in a broad range of biological phenomena. A large variety of glycan structures can be synthesized depending on tissue or cell types and environmental changes. Here, we developed a comprehensive glycosylation mapping tool, termed GlycoMaple, to visualize and estimate glycan structures based on gene expression. We informatically selected 950 genes involved in glycosylation and its regulation. Expression profiles of these genes were mapped onto global glycan metabolic pathways to predict glycan structures, which were confirmed using glycomic analyses. Based on the predictions of N-glycan processing, we constructed 40 knockout HEK293 cell lines and analyzed the effects of gene knockout on glycan structures. Finally, the glycan structures of 64 cell lines, 37 tissues, and primary colon tumor tissues were estimated and compared using publicly available databases. Our systematic approach can accelerate glycan analyses and engineering in mammalian cells.

Selecting cells expressing high levels of recombinant proteins using the GPI-anchored protein with selenocysteine system.

Most biopharmaceutical proteins are produced in mammalian cells because they have the advantageous capacity for protein folding, assembly, and posttranslational modifications. To satisfy the increasing demand for these proteins for clinical purposes and studies, traditional methods to improve protein productivity have included gene amplification, host cell engineering, medium optimization, and screening methods. However, screening and selection of high-producing cell lines remain complex and time consuming. In this study, we established a glycosylphosphatidylinositol (GPI)-anchored protein with a selenocysteine (GPS) system to select cells producing high levels of target secretory proteins. Recombinant lysosomal acid lipase (LIPA) and α-galactosidase A (GALA) were fused with a GPI attachment signal sequence and a selenocysteine insertion sequence after an in-frame UGA codon. Under these conditions, most of the recombinant proteins were secreted into the culture medium, but some were found to be GPI-anchored proteins on the cell surface. When sodium selenite was supplied into the culture medium, the amount of GPI-anchored LIPA and GALA was increased. High-expressing cells were selected by detecting surface GPI-anchored LIPA. The GPI-anchored protein was then eliminated by knocking out the GPI biosynthesis gene PIGK, in these cells, all LIPA was in secreted form. Our system provides a promising method of isolating cells that highly express recombinant proteins from large cell populations.

Calnexin mediates the maturation of GPI-anchors through ER retention.

The protein folding and lipid moiety status of glycosylphosphatidylinositol-anchored proteins (GPI-APs) are monitored in the endoplasmic reticulum (ER), with calnexin playing dual roles in the maturation of GPI-APs. In the present study, we investigated the functions of calnexin in the quality control and lipid remodeling of GPI-APs in the ER. By directly binding the N-glycan on proteins, calnexin was observed to efficiently retain GPI-APs in the ER until they were correctly folded. In addition, sufficient ER retention time was crucial for GPI-inositol deacylation, which is mediated by post-GPI attachment protein 1 (PGAP1). Once the calnexin/calreticulin cycle was disrupted, misfolded and inositol-acylated GPI-APs could not be retained in the ER and were exposed on the plasma membrane. In calnexin/calreticulin-deficient cells, endogenous GPI-anchored alkaline phosphatase was expressed on the cell surface, but its activity was significantly decreased. ER stress induced surface expression of misfolded GPI-APs, but proper GPI-inositol deacylation occurred due to the extended time that they were retained in the ER. Our results indicate that calnexin-mediated ER quality control systems for GPI-APs are necessary for both protein folding and GPI-inositol deacylation.