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BACKGROUND: Acidic lipases with high catalytic activities under acidic conditions have important application values in the food, feed and pharmaceutical industries. However, the availability of acidic lipases is still the main obstacle to their industrial applications. Although a novel acidic lipase Rasamsonia emersonii (LIPR) was heterologously expressed in Escherichia coli, the expression level was unsatisfactory. RESULTS: To achieve the high‐efficiency expression and secretion of LIPR in Pichia pastoris GS115, the combinatorial optimization strategy was adopted including gene codon preference, signal peptide, molecular chaperone co‐expression and disruption of vacuolar sorting receptor VPS10. The activity of the combinatorial optimization engineered strain in a shake flask reached 1480 U mL−1, which was 8.13 times greater than the P. pastoris GS115 parental strain. After high‐density fermentation in a 5‐L bioreactor, the highest enzyme activity reached as high as 11 820 U mL−1. LIPR showed the highest activity at 40 °C and pH 4.0 in the presence of Ca2+ ion. LIPR exhibited strong tolerance to methanol, indicating its potential application in biodiesel biosynthesis. Moreover, the gastrointestinal digestion simulation results demonstrated that LIPR was tolerant to pepsin and trypsin, but its activity was inhibited by sodium taurodeoxycholate. CONCLUSION: This study provided an effective approach for the high expression of acidic lipase LIPR. LIPR was more appropriate for lipid digestion in the stomach than in intestine according to the gastrointestinal digestion simulation results. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

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-β1,3- 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. [ABSTRACT FROM AUTHOR]

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. Whether Rft1 plays a role during M5GN2-PP-Dol translocation has been controversial for over two decades. In this work, a reconstituted in vitro assay demonstrates that purified Rft1 is sufficient to flip M5GN2-PP-Dol across the lipid bilayer. [ABSTRACT FROM AUTHOR]

Since different quantities of white blood cells (WBCs) in solution possess an adaptive osmotic pressure of cells, the WBCs themselves and in solution have similar concentrations, resulting in them having similar dielectric properties. Therefore, a microwave sensor could have difficulty in sensing the quantity variation when WBCs are in solution. This paper presents a highly sensitive, linear permittivity-inspired microwave biosensor for WBCs, counting through the evaporation method. Such a measurement method is proposed to record measurements after the cell solution is dripped onto the chip and is completely evaporated naturally. The proposed biosensor consists of an air-bridged asymmetric differential inductor and a centrally located circular fork-finger capacitor fabricated on a GaAs substrate using integrated passive fabrication technology. It is optimized to feature a larger sensitive area and improved Q-factor, which increases the effective area of interaction between cells and the electromagnetic field and facilitates the detection of their changes in number. The sensing relies on the dielectric properties of the cells and the change in the dielectric constant for different concentrations, and the change in resonance properties, which mainly represents the frequency shift, corresponds to the macroscopic change in the concentration of the cells. The microwave biosensors are used to measure biological samples with concentrations ranging from 0.25 × 106 to 8 × 106 cells per mL in a temperature (26.00 ± 0.40 °C) and humidity (54.40 ± 3.90 RH%) environment. The measurement results show a high sensitivity of 25.06 Hz/cells·mL−1 with a highly linear response of r2 = 0.99748. In addition, a mathematical modeling of individual cells in suspension is performed to estimate the dielectric constant of individual cells and further explain the working mechanism of the proposed microwave biosensor. [ABSTRACT FROM AUTHOR]

Bacterial outer membrane vesicles (OMVs) are potent immunostimulants of regulating the tumor microenvironment (TME) for immunotherapy, and can be used to deliver drugs. However, the severe systemic inflammatory response triggered by OMVs upon intravenous (i.v.) injection has limited their application. Here, we developed a safe and effective strategy by conjugating doxorubicin-loaded serum albumin (SA-DOX, AD) onto the surface of OMVs using a matrix metalloproteinase (MMP)-cleavable peptide linker (cL). This approach enabled the dynamic shielding of OMVs to reduce the systemic side effects while simultaneously enhancing the anti-tumor effects through chemo-immunotherapy. Specifically, the resulting OMV-cL-AD formulation exhibited significantly enhanced accumulation at the tumor site after i.v. administration, facilitated by the SA decoration on the OMVs surface. Subsequently, the shield on the OMV-cL-AD was cleaved by the over-expressed MMP in the TME, leading to the release of both OMVs and AD. This process provided OMV-induced immunotherapy and DOX-induced chemotherapy, resulting in synergistic tumor inhibition. In conclusion, our work demonstrated the potential of OMV-cL-AD as an effective immunochemotherapy strategy that can prolong the survival time of mice without inducing side effects. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Congenital disorders of glycosylation (CDGs) are genetic diseases caused by gene defects in glycan biosynthesis pathways, and there is an increasing number of patients diagnosed with CDGs. Because CDGs show many different clinical symptoms, their accurate clinical diagnosis is challenging. Recently, we have shown that liposome nanoparticles bearing the ALG1-CDG and PMM2-CDG biomarkers (a tetrasaccharide: Neu5Ac-α2,6-Gal-β1,4-GlcNAc-β1,4-GlcNAc) stimulate a moderate immune response, while the generated antibodies show relatively weak affinity maturation. Thus, mature antibodies with class switching to IgG are desired to develop high-affinity antibodies that may be applied in medical applications. Results: In the present study, a liposome-based vaccine platform carrying a chemoenzymatic synthesized phytanyl-linked tetrasaccharide biomarker was optimized. The liposome nanoparticles were constructed by dioleoylphosphatidylcholine (DOPC) to improve the stability and immunogenicity of the vaccine, and adjuvanted with the NKT cell agonist PBS57 to generate high level of IgG antibodies. The results indicated that the reformulated liposomal vaccine stimulated a stronger immune response, and PBS57 successfully induce an antibody class switch to IgG. Further analyses of IgG antibodies elicited by liposome vaccines suggested their specific binding to tetrasaccharide biomarkers, which were mainly IgG2b isotypes. Conclusions: Immunization with a liposome vaccine carrying a carbohydrate antigen and PBS57 stimulates high titers of CDG biomarker-specific IgG antibodies, thereby showing great potential as a platform to develop rapid diagnostic methods for ALG1-CDG and PMM2-CDG. [ABSTRACT FROM AUTHOR]

Immunotherapies are a promising new class of anticancer treatments, but the immunosuppressive tumor microenvironment (TME) hinders their broader implementation. Here, we designed a '3C' strategy based on the conventional drug lentinan (LNT), applying the convertible material polylactic acid with controlled release of LNT (LNT@Mic). Our findings revealed that LNT@Mic exhibited effective biocompatibility coupled with controlled long-term release of LNT. Due to these characteristics, LNT@Mic reprogramed the immunosuppressive TME and demonstrated substantial antitumor activity in the MC38 tumor model. Furthermore, it served as a facile and generalizable cancer immunotherapy strategy for augmenting LNT bioavailability while enhancing the efficacy of anti-programmed death-ligand 1 therapy against the 'cold' 4T1 tumor model. These findings provide a reference for tumor immunotherapy strategies for the further study and application of LNT. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: The Siglec-Fc protein, a fusion protein combining Siglec with the Fc part of a human antibody, is a promising sialic acid-Siglec axis-targeted agent for cancer treatment and is widely used for Siglec ligands discovery. The recombinant Siglec-Fc fusion protein has been expressed in different cell systems. However, its characteristics have not been investigated in detail. In this study, HEK293 and CHO cell lines were used to express the Siglec9-Fc protein, and their adaptability for production was compared. We optimized culture conditions and compared the glycosylation, yield, dimerization and sialic acid binding activity of the Siglec9-Fc protein produced in HEK293 and CHO. Using purified recombinant protein, we further analyzed the distribution of Siglec9 ligands on cancer cell lines, as well as bladder cancer tissue, and revealed the potential ligands. Our findings provide support for the selection of Siglec9-Fc protein expression systems and detection of related Siglec9 ligands. The emerging importance of the Siglec-sialic acid axis in human disease, especially cancer, has necessitated the identification of ligands for Siglecs. Recombinant Siglec-Fc fusion proteins have been widely used as ligand detectors, and also as sialic acid-targeted antibody-like proteins for cancer treatment. However, the heterogenetic properties of the Siglec-Fc fusion proteins prepared from various expression systems have not been fully elucidated. In this study, we selected HEK293 and CHO cells for producing Siglec9-Fc and further evaluated the properties of the products. The protein yield in CHO (8.23 mg/L) was slightly higher than that in HEK293 (7.46 mg/L). The Siglec9-Fc possesses five N-glycosylation sites and one of them is located in its Fc domain, which is important for the quality control of protein production and also the immunogenicity of Siglec-Fc. Our glycol-analysis confirmed that the recombinant protein from HEK293 received more fucosylation, while CHO showed more sialylation. Both products revealed a high dimerization ratio and sialic acid binding activity, which was confirmed by the staining of cancer cell lines and bladder cancer tissue. Finally, our Siglec9-Fc product was used to analyze the potential ligands on cancer cell lines. [ABSTRACT FROM AUTHOR]

Rare sugars are monosaccharides with low natural abundance. They are structural isomers of dietary sugars, but hardly be metabolized. Here, we report that rare sugar l-sorbose induces apoptosis in various cancer cells. As a C-3 epimer of d-fructose, l-sorbose is internalized via the transporter GLUT5 and phosphorylated by ketohexokinase (KHK) to produce l-sorbose-1-phosphate (S-1-P). Cellular S-1-P inactivates the glycolytic enzyme hexokinase resulting in attenuated glycolysis. Consequently, mitochondrial function is impaired and reactive oxygen species are produced. Moreover, l-sorbose downregulates the transcription of KHK-A, a splicing variant of KHK. Since KHK-A is a positive inducer of antioxidation genes, the antioxidant defense mechanism in cancer cells can be attenuated by l-sorbose-treatment. Thus, l-sorbose performs multiple anticancer activities to induce cell apoptosis. In mouse xenograft models, l-sorbose enhances the effect of tumor chemotherapy in combination with other anticancer drugs. These results demonstrate l-sorbose as an attractive therapeutic reagent for cancer treatment. The rare sugar L-sorbose is shown to decrease cell viability and increase apoptotic cells in culture and to enhance the effect of tumor chemotherapy in combination with sorafenib in mouse xenograft models. [ABSTRACT FROM AUTHOR]

A crack-free AlN film with 4.5 μm thickness was grown on a 2-inch hole-type nano-patterned sapphire substrates (NPSSs) by hydride vapor phase epitaxy (HVPE). The coalescence, stress evolution, and dislocation annihilation mechanisms in the AlN layer have been investigated. The large voids located on the pattern region were caused by the undesirable parasitic crystallites grown on the sidewalls of the nano-pattern in the early growth stage. The coalescence of the c-plane AlN was hindered by these three-fold crystallites and the special triangle void appeared. The cross-sectional Raman line scan was used to characterize the change of stress with film thickness, which corresponds to the characteristics of different growth stages of AlN. Threading dislocations (TDs) mainly originate from the boundary between misaligned crystallites and the c-plane AlN and the coalescence of two adjacent c-plane AlN crystals, rather than the interface between sapphire and AlN. [ABSTRACT FROM AUTHOR]

BACKGROUND: Rare sugars have become promising 'sugar alternatives' because of their low calories and unique physiological functions. Among the family of rare sugars, d‐allulose is one of the sugars attracting interest. Ketose 3‐epimerases (KEase), including d‐tagatose 3‐epimerase (DTEase) and d‐allulose 3‐epimerase (DAEase), are mainly used for d‐allulose production. RESULTS: In this study, a putative xylose isomerase from Caballeronia insecticola was characterized and identified as a novel DAEase. Caballeronia insecticola DAEase displayed prominent enzymatic properties, and 150 g L−1d‐allulose was produced from 500 g L−1d‐fructose in 45 min with a conversion rate of 30% and high productivity of 200 g L−1 h−1. Furthermore, DAEase was employed in a phosphorylation–dephosphorylation cascade reaction, which significantly increased the conversion rate of d‐allulose. Under optimized conditions, the conversion rate of d‐allulose was approximately 100% when the concentration of d‐fructose was 50 mmol L−1. CONCLUSION: This research described a very beneficial and facile approach for d‐allulose production based on C. insecticola DAEase. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

In mammals, both professional phagocytes and nonprofessional phagocytes (NPPs) can perform phagocytosis. However, limited targets are phagocytosed by NPPs, and thus, the mechanism remains unclear. We find that spores of the yeast Saccharomyces cerevisiae are internalized efficiently by NPPs. Analyses of this phenomenon reveals that RNA fragments derived from cytosolic RNA species are attached to the spore wall, and these fragments serve as ligands to induce spore internalization. Furthermore, we show that a multiligand receptor, RAGE (receptor for advanced glycation end-products), mediates phagocytosis in NPPs. RAGE-mediated phagocytosis is not uniquely induced by spores but is an intrinsic mechanism by which NPPs internalize macromolecules containing RAGE ligands. In fact, artificial particles labeled with polynucleotides, HMGB1, or histone (but not bovine serum albumin) are internalized in NPPs. Our findings provide insight into the molecular basis of phagocytosis by NPPs, a process by which a variety of macromolecules are targeted for internalization. The multiligand receptor RAGE (receptor for advanced glycation end-products) mediates phagocytosis in non-professional phagocytes (NPPs), for example through the use of RNA fragments as ligands for internalization. [ABSTRACT FROM AUTHOR]

Glycerol is a potential sustainable feedstock, and biorefining processes to convert glycerol into value-added chemicals have been developed over the past decade. Alditol oxidase (AldO) is capable of selectively oxidizing the primary hydroxyl groups of alditols such as glycerol. In this study, a new FAD-binding protein from Thermopolyspora flexuosa was expressed and identified as a novel alditol oxidase (AldOT. fle). AldOT. fle displayed the optimal activity at pH 8.0 and 25 °C. AldOT. fle was not metal-dependent, but the activity was completely inhibited by Fe3+. AldOT. fle had a wide substrate specificity and high catalytic efficiency for glycerol. Furthermore, the recombinant AldOT. fle could produce d-glyceric acid from glycerol with a conversion rate ranging from 86.6% (5 mM glycerol) to 20.5% (500 mM glycerol). The recombinant E. coli with AldOT. fle could also produce 23.8 mM d-glyceric acid from 100 mM glycerol. The recombinant AldOT. fle had the potential to produce other aldehyde products by selectively oxidizing the hydroxyl groups of alditols and many other commodity chemicals by redesigning glycerol metabolism. [ABSTRACT FROM AUTHOR]

To increase antibody secretion and dose sparing, squalene-in-water aluminium hydrogel (alum)-stabilised emulsions (ASEs) have been developed, which offer increased surface areas and cellular interactions for higher antigen loading and enhanced immune responses. Nevertheless, the squalene (oil) in previous attempts suffered from limited oxidation resistance, thus, safety and stability were compromised. From a clinical translational perspective, it is imperative to screen the optimal oils for enhanced emulsion adjuvants. Here, because of the varying oleic to linoleic acid ratio, soybean oil, peanut oil, and olive oil were utilised as oil phases in the preparation of aluminium hydrogel-stabilised squalene-in-water emulsions, which were then screened for their stability and immunogenicity. Additionally, the underlying mechanisms of oil phases and emulsion stability were unravelled, which showed that a higher oleic to linoleic acid ratio increased anti-oxidative capabilities but reduced the long-term storage stability owing to the relatively low zeta potential of the prepared droplets. As a result, compared with squalene-in-water ASEs, soybean-in-water ASEs exhibited comparable immune responses and enhanced stability. By optimising the oil phase of the emulsion adjuvants, this work may offer an alternative strategy for safe, stable, and effective emulsion adjuvants. [ABSTRACT FROM AUTHOR]

D-Allulose, a generally regarded as safe (GRAS) sugar, is rare in nature. It is among the most promising sweeteners for future use due to its low caloric content, sucrose-like taste, and unique functions. D-Allulose has many physiological effects, such as antiobesity, antihyperglycemia, antidiabetes, anti-inflammatory, antioxidant, and neuroprotective effects. Therefore, D-allulose has important application value in the food, pharmaceutical, and healthcare industries. However, the high cost of D-allulose production limits its large-scale application. Currently, biotransformation is very attractive for D-allulose synthesis, with the two main methods of biosynthesis being the Izumoring strategy and the DHAP-dependent aldolase strategy. This article reviews recent advances regarding the physiological functions and biosynthesis of D-allulose. In addition, future perspectives on the production of D-allulose are presented. [ABSTRACT FROM AUTHOR]

N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER). Alg2 mannosyltransferase adds both the α1,3- and α1,6-mannose (Man) onto ManGlcNAc2-pyrophosphate-dolichol (M1Gn2-PDol) in either order to generate the branched M3Gn2-PDol product. The well-studied yeast Alg2 interacts with ER membrane through four hydrophobic domains. Unexpectedly, we show that Alg2 structure has diverged between yeast and humans. Human Alg2 (hAlg2) associates with the ER via a single membrane-binding domain and is markedly more stable in vitro. These properties were exploited to develop a liquid chromatography-mass spectrometry quantitative kinetics assay for studying purified hAlg2. Under physiological conditions, hAlg2 prefers to transfer α1,3-Man onto M1Gn2 before adding the α1,6-Man. However, this bias is altered by an excess of GDP-Man donor or an increased level of M1Gn2 substrate, both of which trigger production of the M2Gn2(α-1,6)-PDol. These results suggest that Alg2 may regulate the LLO biosynthetic pathway by controlling accumulation of M2Gn2 (α-1,6) intermediate. Despite the conservation of N-glycosylation, human and yeast Alg2 structures have diverged with distinct ER-binding topologies. The human enzyme is more stable than the yeast orthologue, and its activity is modulated by the concentration of donor or acceptor substrate. [ABSTRACT FROM AUTHOR]

Vps13 family proteins are proposed to function in bulk lipid transfer between membranes, but little is known about their regulation. During sporulation of Saccharomyces cerevisiae, Vps13 localizes to the prospore membrane (PSM) via the Spo71–Spo73 adaptor complex. We previously reported that loss of any of these proteins causes PSM extension and subsequent sporulation defects, yet their precise function remains unclear. Here, we performed a genetic screen and identified genes coding for a fragment of phosphatidylinositol (PI) 4-kinase catalytic subunit and PI 4-kinase noncatalytic subunit as multicopy suppressors of spo73Δ. Further genetic and cytological analyses revealed that lowering PI4P levels in the PSM rescues the spo73Δ defects. Furthermore, overexpression of VPS13 and lowering PI4P levels synergistically rescued the defect of a spo71Δ spo73Δ double mutant, suggesting that PI4P might regulate Vps13 function. In addition, we show that an N-terminal fragment of Vps13 has affinity for the endoplasmic reticulum (ER), and ER-plasma membrane (PM) tethers localize along the PSM in a manner dependent on Vps13 and the adaptor complex. These observations suggest that Vps13 and the adaptor complex recruit ER-PM tethers to ER-PSM contact sites. Our analysis revealed that involvement of a phosphoinositide, PI4P, in regulation of Vps13, and also suggest that distinct contact site proteins function cooperatively to promote de novo membrane formation. Author summary: Vps13 family proteins are conserved lipid transfer proteins that function at organelle contact sites and have been implicated in a number of different neurological diseases. In the yeast Saccharomyces cerevisiae, Vps13 is encoded by a single gene and is localized to various contact sites by interaction with different adaptor proteins and/or lipids, however its regulation is yet to be clarified. We have previously shown that during the developmental process of sporulation, Vps13 is recruited to de novo membrane structures called prospore membranes (PSMs) by a specific adaptor complex, and Vps13 and its adaptors are required for PSM extension. Here we reveal that loss of an adaptor can be overcome by lowering phosphatidylinositol-4-phosphate (PI4P) levels, either by inhibiting PI 4-kinase on the PSM or recruiting PI 4-phospatase to the PSM and that PI4P levels in the PSM affect Vps13 function. Further, we show that Vps13 forms endoplasmic reticulum (ER)-PSM contact sites, that ER-plasma membrane tethering proteins are recruited to ER-PSM contacts, and these proteins may function in conjunction with Vps13. Thus, our work shines light on both the mechanisms of intracellular remodeling and the function of this important class of lipid transfer proteins. [ABSTRACT FROM AUTHOR]

Glycoprotein therapeutics are among the leading products in the biopharmaceutical industry. The heterogeneity of glycans in therapeutic proteins is an issue for maintaining quality, activity and safety during bioprocessing. In this study, we knocked out genes encoding Golgi α-mannosidase-II, MAN2A1 and MAN2A2 in human embryonic kidney 293 (HEK293) cells, establishing an M2D-KO cell line that can produce recombinant proteins mainly with hybrid-type N-glycans. Furthermore, FUT8 , which encodes α1,6-fucosyltransferase, was knocked out in the M2D-KO cell line, establishing a DF-KO cell line that can express noncore fucosylated hybrid-type N-glycans. Two recombinant proteins, lysosomal acid lipase and constant fragment of human IgG1, were expressed in the M2D-KO and DF-KO cell lines. Glycan structural analysis revealed that complex-type N-glycans were removed in both M2D-KO and DF-KO cells. Our results suggest that these cell lines are suitable for the production of therapeutic proteins with hybrid-type N-glycans. Moreover, KO cell lines would be useful as models for researching the mechanism of antimetastatic effects in human tumours by swainsonine treatment. [ABSTRACT FROM AUTHOR]

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. Liu et al. construct a knock out cell library of genes involved in the biosynthesis of glycosylphosphatidylinositol (GPI)-anchored protein (GPI-APs) from HEK-293 cells and analyzed the surface expression and sensitivity to phosphatidylinositol-specific phospholipase C. Furthermore, the authors also identify structural motifs of GPIs that are recognized by a GPI-binding toxin, aerolysin. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This work focused on the separation of the active ingredients of maca (Lepidium meyenii Walpers) and evaluated the antioxidative capability of these components with effects on improving glucose and lipid metabolism in insulin‐resistant HepG2 cells. DPPH free radical scavenging and reducing power assays were used to evaluate the antioxidant activity of maca extracts. An insulin‐resistant HepG2 cell model induced by glucose, fructose, oleic acid, and palmitic acid was adopted to investigate the effects of maca extracts on regulating glucose and lipid metabolism in this study. LC‐MS/MS was then used for determination of the maca n‐butanol (NBT) subfraction. The results showed that maca ethanol extract and subfractions of this extract exhibited certain antioxidant capacity. Furthermore, the NBT subfraction reversed the disorders in glucose and lipid metabolism in insulin‐resistant HepG2 cells and significantly increased the mRNA expression of phosphoinositide 3‐kinases (PI3K) and AKT in insulin‐resistant HepG2 cells in a dose‐dependent manner. In addition, the LC‐MS/MS results showed that the NBT subfraction contained many active ingredients. Overall, this study suggests that the NBT subfraction of the ethanol extract rich in glucosinolates modulates insulin resistance via PI3K/AKT activation in insulin‐resistant HepG2 cells and might exert potentially beneficial effects in improving or treating glucose and lipid metabolic disorders. [ABSTRACT FROM AUTHOR]

The positron emission tomography (PET) could predict the prognosis of DLBCL patients, but the exact procedure on interim PET (iPET) to determine chemoresistant patients remains elusive. We retrospectively analyzed 593 newly diagnosed DLBCL patients uniformly treated with R-CHOP regimen. Among them, 352 patients diagnosed from August 2010 to December 2016 were included in the test cohort and 241 patients diagnosed from January 2017 to December 2019 were included in the validation cohort. The iPET was evaluated with Deauville criteria and ΔSUVmax method. The reduction of maximal SUV between baseline and after 4 cycles of chemotherapy were defined as ΔSUVmax. The survival functions were depicted using the Kaplan–Meier method and compared with the log-rank test. Patients with iPET Deauville 4 had heterogeneous outcome and end of treatment complete response rates (eCRR). Combined Deauville with ΔSUVmax method, we proposed a modified-Deauville model: patients with Deauville 4 and ΔSUVmax > 70%, as well as those with Deauville 1–3, were reclassified into the modified-Deauville negative group, while patients with Deauville 4 and ΔSUVmax ≤ 70%, as well as those with Deauville 5, into the modified-Deauville positive group. In the test cohort, 3-year PFS, OS and eCRR of modified-Deauville negative group were 80.2%, 89.9% and 91.8%, significantly higher than those of positive group (12.5%, 27.3% and 29.2%, p ≤.001). Similar results were found in the validation cohort, that 3-year PFS, OS and eCRR were 87.8%, 95.4%, 96.3% in modified-Deauville negative group, and 27.4%, 32.5%, 13.5% in positive group. Through modified-Deauville model, patients in iPET positive group had very low eCRR and were resistant to conventional chemotherapy. The modified-Deauville model could better distinguish DLBCL patients with poor response to chemotherapy. Accordingly, these patients could be recognized early and provided with alternative therapeutic agents, which might improve the clinical outcome of refractory DLBCL patients. [ABSTRACT FROM AUTHOR]

A FLAG tag consisting of DYKDDDDK is an epitope tag that is frequently and widely used to detect recombinant proteins of interest. In this study, we performed a CRISPR-based genetic screening to identify factors involved in the detection of a FLAG-tagged misfolded model protein at the cell surface. In the screening, SLC35B2, which encodes 3'-phosphoadenosine-5'-phosphosulfate transporter 1, was identified as the candidate gene. The detection of FLAG-tagged misfolded proteins at the cell surface was significantly increased in SLC35B2-knockout cells. Furthermore, protein tyrosine sulfation mediated by tyrosyl-protein sulfotransferase 2 (TPST2) suppressed FLAG-tagged protein detection. Localization analysis of the FLAG-tagged misfolded proteins confirmed that defects in tyrosine sulfation are only responsible for enhancing anti-FLAG staining on the plasma membrane but not inducing the localization change of misfolded proteins on the plasma membrane. These results suggest that a FLAG tag on the misfolded protein would be sulfated, causing a reduced detection by the M2 anti-FLAG antibody. Attention should be required when quantifying the FLAG-tagged proteins in the secretory pathway. [ABSTRACT FROM AUTHOR]

Objective * To investigate the activity detection method of α-1,3-mannosyltransferase (ALG3) mutant protein in congenital disorder of glycosylation (ALG3-CDG), and examine the relationship between the activity and the severity of disease. Methods * The mutation sites of the patients in the reports were I69T, W71R, G96R, M157K, R171Q and M209T, respectively. After homologous alignment of the amino acid sequences of Homo sapiens and Saccharomyces cerevisiae (S. cerevisiae) ALG3, six yeast mutant proteins (I70T, Y72R, G98R, L157K, R171Q and M221T) related to ALG3-CDG were designed. These mutants were expressed in Escherichia coli (E. coli). Using E. coli membrane containing wild-type ALG3 or its mutant proteins as catalysts, the relative activities of mutant proteins were calculated by liquid chromatography tandem mass spectrometry based on the catalytic activity of wild type protein. The activities of corresponding mutations in the patients with different survival time were compared. Results * Six ALG3-CDG related S. cerevisiae conserved ALG3 mutant proteins were expressed in E. coli in the comparable protein expression levels with wild type ALG3. The in vitro quantitative activity assay of enzymes showed that compared with the wild type ALG3, the relative activities of the mutant proteins I70T, Y72R, G98R, L157K, R171Q and M221T were 2.8%, 4.9%, 4.5%, 17.2%, 4.8% and 22.3%, respectively. The corresponding mutation activity was significantly higher in the patients with survival longer than 1 year than that in the patients with survival less than 1 year (P=0.002). Conclusion * An in vitro quantitative method is established to measure the enzymatic activities of ALG3-CDG related S. cerevisiae ALG3 mutants, providing the possibility to predict the severity of ALG3-CDG. [ABSTRACT FROM AUTHOR]

In the budding yeast Saccharomyces cerevisiae, Svl3 and Pam1 proteins work as functional homologues. Loss of their function causes increased levels of chitin deposition in the cell wall and temperature sensitivity, suggesting their involvement in cell wall formation. We found that the N‐ and C‐termini of these proteins have distinctive and critical functions. They contain an N‐terminal part that has a probable 2‐dehydropantoate 2‐reductase domain. In Svl3, this part can be replaced with the yeast 2‐dehydropantoate 2‐reductase, Pan5, suggesting that Svl3 and its homologues may be able to mediate 2‐dehydropantoate 2‐reductase function. On the other hand, Svl3 is recruited to the bud tip and bud neck via multiple localization signals in the C‐terminal part. One of such signals is the lysine‐rich region located in the C‐terminal end. The function and localization of Svl3 are significantly disrupted by the loss of this lysine‐rich region; however, its localization is not completely abolished by the mutation because another localization signal enables appropriate transport. Svl3 and Pam1 orthologues are found in cells across fungal species. The Svl3 orthologues of Candida glabrata can complement the loss of Svl3 and Pam1 in S. cerevisiae. C. glabrata cells lacking the SVL3 and PAM1 orthologue genes exhibit phenotypes similar to those observed in svl3∆pam1∆ S. cerevisiae cells. Thus, Svl3 homologues may be generally required for the assembly of the cell wall in fungal cells. [ABSTRACT FROM AUTHOR]

Therapeutic proteins are a developing part of the modern biopharmaceutical industry, providing novel therapies to intractable diseases including cancers and autoimmune diseases. The human embryonic kidney 293 (HEK293) cell line has been widely used to produce recombinant proteins in both basic science and industry. The heterogeneity of glycan structures is one of the most challenging issues in the production of therapeutic proteins. Previously, we knocked out genes encoding α1,2-mannosidase-Is, MAN1A1 , MAN1A2 and MAN1B1 , in HEK293 cells, establishing a triple-knockout (T-KO) cell line, which produced recombinant protein with mainly high-mannose-type N-glycans. Here, we further knocked out MAN1C1 and MGAT1 encoding another Golgi α1,2-mannosidase-I and N-acetylglucosaminyltransferase-I, respectively, based on the T-KO cells. Two recombinant proteins, lysosomal acid lipase (LIPA) and immunoglobulin G1 (IgG1), were expressed in the quadruple-KO (QD-KO) and quintuple-KO (QT-KO) cell lines. Glycan structural analysis revealed that all the hybrid-type and complex-type N-glycans were eliminated, and only the high-mannose-type N-glycans were detected among the recombinant proteins prepared from the QD-KO and QT-KO cells. Overexpression of the oncogenes MYC and MYCN recovered the slow growth in QD-KO and QT-KO without changing the glycan structures. Our results suggest that these cell lines could be suitable platforms to produce homogeneous therapeutic proteins. [ABSTRACT FROM AUTHOR]

In eukaryotes, the biosynthesis of a highly conserved dolichol-linked oligosaccharide (DLO) precursor Glc3Man9GlcNAc2-pyrophosphate-dolichol (PP-Dol) begins on the cytoplasmic face of the endoplasmic reticulum (ER) and ends within the lumen. Two functionally distinguished heteromeric glycosyltransferase (GTase) complexes are responsible for the cytosolic DLO assembly. Alg1, a β-1, 4 mannosyltransferase (MTase) physically interacts with Alg2 and Alg11 proteins to form the multienzyme complex which catalyzes the addition of all five mannose to generate the Man5GlcNAc2-PP-Dol intermediate. Despite the fact that Alg1 plays a central role in the formation of the multi-MTase has been confirmed, the topological information of Alg1 including the molecular mechanism of membrane association are still poorly understood. Using a combination of bioinformatics and biological approaches, we have undertaken a structural and functional study on Alg1 protein, in which the enzymatic activities of Alg1 and its variants were monitored by a complementation assay using the GALpr - ALG1 yeast strain, and further confirmed by a liquid chromatography–mass spectrometry-based in vitro quantitative assay. Computational and experimental evidence confirmed Alg1 shares structure similarity with Alg13/14 complex, which has been defined as a membrane-associated GT-B GTase. Particularly, we provide clear evidence that the N-terminal transmembrane domain including the following positively charged amino acids and an N-terminal amphiphilic-like α helix domain exposed on the protein surface strictly coordinate the Alg1 orientation on the ER membrane. This work provides detailed membrane topology of Alg1 and further reveals its biological importance at the spatial aspect in coordination of cytosolic DLO biosynthesis. [ABSTRACT FROM AUTHOR]

Oligosaccharyltransferases (OSTs) mediate the en bloc transfer of N‐glycan intermediates onto the asparagine residue in glycosylation sequons (N‐X‐S/T, X≠P). These enzymes are typically heteromeric complexes composed of several membrane‐associated subunits, in which STT3 is highly conserved as a catalytic core. Metazoan organisms encode two STT3 genes (STT3A and STT3B) in their genome, resulting in the formation of at least two distinct OST isoforms consisting of shared subunits and complex specific subunits. The STT3A isoform of OST primarily glycosylates substrate polypeptides cotranslationally, whereas the STT3B isoform is involved in cotranslational and post‐translocational glycosylation of sequons that are skipped by the STT3A isoform. Here, we describe mutant constructs of monomeric enhanced green fluorescent protein (mEGFP), which are susceptible to STT3B‐dependent N‐glycosylation. The endoplasmic reticulum‐localized mEGFP (ER‐mEGFP) mutants contained an N‐glycosylation sequon at their C‐terminus and exhibited increased fluorescence in response to N‐glycosylation. Isoform‐specific glycosylation of the constructs was confirmed by using STT3A‐ or STT3B‐knockout cell lines. Among the mutant constructs that we tested, the ER‐mEGFP mutant containing the N185‐C186‐T187 sequon was the best substrate for the STT3B isoform in terms of glycosylation efficiency and fluorescence change. Our results suggest that the mutant ER‐mEGFP is useful for monitoring STT3B‐dependent post‐translocational N‐glycosylation in cells of interest, such as those from putative patients with a congenital disorder of glycosylation. [ABSTRACT FROM AUTHOR]

Abstract: In eukaryotes, the glycosylphosphatidylinositol (GPI) modification of many glycoproteins on the cell surface is highly conserved. The lipid moieties of GPI‐anchored proteins undergo remodelling processes during their maturation. To date, the products of the PER1, GUP1 and CWH43 genes of the yeast Saccharomyces cerevisiae have been shown to be involved in the lipid remodelling. Here, we focus on the putative GPI remodelling pathway in the methylotrophic yeast Ogataea minuta. We found that the O. minuta homologues of PER1, GUP1 and CWH43 are functionally compatible with those of S. cerevisiae. Disruption of GUP1 or CWH43 in O. minuta caused a growth defect under non‐permissive conditions. The O. minuta per1Δ mutant exhibited a more fragile phenotype than the gup1Δ or cwh43Δ mutants. To address the role of GPI modification in O. minuta, we assessed the effect of these mutations on the processing and localization of the O. minuta homologues of the Gas1 protein; in S. cerevisiae, Gas1p is an abundant and well‐characterized GPI‐anchored protein. We found that O. minuta possesses two copies of the GAS1 gene, which we designate GAS1A and GAS1B. Microscopy and western blotting analysis showed mislocalization and/or lower retention of Gas1Ap and Gas1Bp within the membrane fraction in per1Δ or gup1Δ mutant cells, suggesting the significance of lipid remodelling for GPI‐anchored proteins in O. minuta. Localization behaviour of Gas1Bp differed from that of Gas1Ap. Our data reveals, for the first time (to our knowledge), the existence of genes related to GPI anchor remodelling in O. minuta cells. [ABSTRACT FROM AUTHOR]

The yeast spore wall is an excellent model to study the assembly of an extracellular macromolecule structure. In the present study, mutants defective in β-1,6-glucan synthesis, including kre1∆, kre6∆, kre9∆ and big1∆, were sporulated to analyse the effect of β-1,6-glucan defects on the spore wall. Except for kre6∆, these mutant spores were sensitive to treatment with ether, suggesting that the mutations perturb the integrity of the spore wall. Morphologically, the mutant spores were indistinguishable from wild-type spores. They lacked significant sporulation defects partly because the chitosan layer, which covers the glucan layer, compensated for the damage. The proof for this model was obtained from the effect of the additional deletion of CHS3 that resulted in the absence of the chitosan layer. Among the double mutants, the most severe spore wall deficiency was observed in big1∆ spores. The majority of the big1∆chs3∆ mutants failed to form visible spores at a higher temperature. Given that the big1∆ mutation caused a failure to attach a GPI-anchored reporter, Cwp2-GFP, to the spore wall, β-1,6-glucan is involved in tethering of GPI-anchored proteins in the spore wall as well as in the vegetative cell wall. Thus, β-1,6-glucan is required for proper organization of the spore wall. Copyright © 2017 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

All asparagine-linked glycans ( N-glycans) on the eukaryotic glycoproteins are primarily derived from dolichol-linked oligosaccharides (DLO), synthesized on the rough endoplasmic reticulum membrane. We have previously reported cloning and identification of the human gene, HMT- 1, which encodes chitobiosyldiphosphodolichol beta-mannosyltransferase (β1,4-MT) involved in the early assembly of DLO. Considering that N-glycosylation is one of the most ubiquitous post-translational modifications for many eukaryotic proteins, the HMT- 1 could be postulated as one of the housekeeping genes, but its transcriptional regulation remains to be investigated. Here we screened a 1 kb region upstream from HMT- 1 open reading frame (ORF) for transcriptionally regulatory sequences by using chloramphenicol acetyl transferase (CAT) assay, and found that the region from −33 to −1 positions might act in HMT- 1 transcription at basal level and that the region from −200 to −42 should regulate its transcription either positively or negatively. In addition, results with CAT assays suggested the possibility that two GATA-1 motifs and an Sp1 motif within a 200 bp region upstream from HMT- 1 ORF might significantly upregulate HMT- 1 transcription. On the contrary, the observations obtained from site-directed mutational analyses revealed that an NF-1/AP-2 overlapping motif located at −148 to −134 positions should serve as a strong silencer. The control of the HMT- 1 transcription by these motifs resided within the 200 bp region could partially explain the variation of expression level among various human tissues, suggesting availability and importance of this region for regulatory role in HMT- 1 expression. [ABSTRACT FROM AUTHOR]

Objectives: To achieve consecutive conversion from creatinine to urea and sarcosine using creatininase and creatinase encapsulated in spores of Saccharomyces cerevisiae. Results: Creatininase encapsulated into the spore wall was produced and its specific activity was 3.4 ± 0.4 U/mg. By deletion of OSW2 gene, which causes a mild spore wall defect, the activity was increased to 10.9 ± 0.5 U/mg. Compared with soluble enzymes, spore-encapsulated creatininase was tolerant to environmental stresses; creatininase encapsulated in osw2∆ spores retained more than 90 % of the activity after treatment by SDS or proteinase K. Creatinase capsules could also be produced through spore encapsulation. The mixture of spores containing either creatininase or creatinase could mediate a two-step reaction to produce urea from creatinine; 5 mg spores produced 19 µmol urea in 10 min. Spores co-expressing creatininase and creatinase could also mediate the reactions more efficiently than the mixture of spores individually expressing each enzyme; the yield in 10 min was 38 µmol. Conclusions: Yeast spores can hold creatininase and creatinase simultaneously and catalyze the consecutive reactions. [ABSTRACT FROM AUTHOR]

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Glycosylphosphatidylinositol (GPI) is synthesized and transferred to proteins in the endoplasmic reticulum (ER). GPI-anchored proteins are then transported from the ER to the plasma membrane through the Golgi apparatus. To date, at least 17 steps have been identified to be required for the GPI biosynthetic pathway. Here, we aimed to establish a comprehensive screening method to identify genes involved in GPI biosynthesis using mammalian haploid screens. Human haploid cells were mutagenized by the integration of gene trap vectors into the genome. Mutagenized cells were then treated with a bacterial pore-forming toxin, aerolysin, which binds to GPI-anchored proteins for targeting to the cell membrane. Cells that showed low surface expression of CD59, a GPI-anchored protein, were further enriched for. Gene trap insertion sites in the non-selected population and in the enriched population were determined by deep sequencing. This screening enriched 23 gene regions among the 26 known GPI biosynthetic genes, which when mutated are expected to decrease the surface expression of GPI-anchored proteins. Our results indicate that the forward genetic approach using haploid cells is a useful and powerful technique to identify factors involved in phenotypes of interest. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae spores are dormant cells, which can tolerate various types of environmental stress. In our previous work, we successfully developed biological and chemical methods for enzyme immobilization based on the structures of S. cerevisiae spore wall. In this study, we employed biological and chemical approaches for the immobilization of d-xylose isomerase (XI) from Thermus thermophilus and d-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens with yeast spores, respectively. The enzymatic properties of both immobilized XI and DPEase were characterized and the immobilized enzymes exhibit higher thermostability, broader pH tolerance, and good repeatability compared with free enzymes. Furthermore, we established a two-step approach for the bioconversion of d-glucose to d-psicose using immobilized enzymes. To improve the conversion yield, a multi-pot strategy was adopted for d-psicose production by repeating the two-step process continually. As a result, the yield of d-psicose was obviously improved and the highest yield reached about 12.0 %. [ABSTRACT FROM AUTHOR]

Gastropulmonary route of infection was considered to be an important mechanism of ventilator-associated pneumonia (VAP). However there is little evidence to support this assumption. Moreover, the prevalence of microaspiration in elderly ventilated patients was not well understood. To confirm gastropulmonary infection route and investigate the prevalence of microaspiration in elderly ventilated patients using genome macrorestriction-pulsed field gel electrophoresis (GM-PFGE). Patients over 60 years old, expected to receive mechanical ventilation longer than 48 h, were prospectively enrolled from October 2009 to January 2012. Clinical data were collected and recorded until they died, developed pneumonia, or were extubated. Samples from gastric fluid, subglottic secretion and lower respiratory tract (LRT) were collected during the follow-up for microbiological examination. To evaluate the homogeneity, GM-PFGE was performed on strains responsible for VAP that had the same biochemical phenotype as those isolated from gastric juice and subglottic secretions sequentially. Among 44 VAP patients, 76 strains were isolated from LRT and considered responsible for VAP. Twenty-two isolates had the same biochemical phenotype with the corresponding gastric isolates. The homology was further confirmed using GM-PFGE in 12 episodes of VAP. Nearly 30 % of VAPs were caused by microaspiration based on the analysis of bacterial phenotype or GM-PFGE. In addition, 58.3 % patients with gastric colonization developed VAP, especially late-onset VAP (LOP). Gastropulmonary infection route exists in VAP especially LOP in elderly ventilated patients. It is one of the important mechanisms in the development of VAP. [ABSTRACT FROM AUTHOR]

Protein N-glycosylation begins with the assembly of a lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER) membrane. The first two steps of LLO biosynthesis are catalyzed by a functional multienzyme complex comprised of the Alg7 GlcNAc phosphotransferase and the heterodimeric Alg13/Alg14 UDP-GlcNAc transferase on the cytosolic face of the ER. In the Alg13/14 glycosyltransferase, Alg14 recruits cytosolic Alg13 to the ER membrane through interaction between their C-termini. Bioinformatic analysis revealed that eukaryotic Alg14 contains an evolved N-terminal region that is missing in bacterial orthologs. Here, we show that this N-terminal region of Saccharomyces cerevisiae Alg14 localize its green fluorescent protein fusion to the ER membrane. Deletion of this region causes defective growth at 38.5°C that can be partially complemented by overexpression of Alg7. Coimmunoprecipitation demonstrated that the N-terminal region of Alg14 is required for direct interaction with Alg7. Our data also show that Alg14 lacking the N-terminal region remains on the ER membrane through a nonperipheral association, suggesting the existence of another membrane-binding site. Mutational studies guided by the 3D structure of Alg14 identified a conserved α-helix involved in the second membrane association site that contributes to an integral interaction and protein stability. We propose a model in which the N- and C-termini of Alg14 coordinate recruitment of catalytic Alg7 and Alg13 to the ER membrane for initiating LLO biosynthesis. [ABSTRACT FROM PUBLISHER]