rapid-glyco-qualitative-assessment-recombinant-proteins-using-fully

Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, ...

National Institute of Advanced Industrial Science and Technology

The core 1 β1,3-galactosyltransferase-specific molecular chaperon (Cosmc) is essential for the synthesis of the core 1 structure of mucin-type O-glycans. To clarify the physiological role of core 1-derived O-glycans in macrophages, we exploited the LysM-Cre transgene to generate a conditional Cosmc mutant allele (conditional Cosmc knockout; cKO) in myeloid cells. cKO mice developed normally with no gross phenotypic abnormalities or abnormal peripheral blood counts. Resident peritoneal macrophages (rpMacs) of cKO mice exhibited impaired engulfment of apoptotic cells but showed normal macrophage differentiation and counts. T-cell immunoglobulin and mucin domain-containing molecule 4 (Tim4) is a phosphatidylserine (PS) receptor expressed on rpMacs and possesses a heavily O-glycosylated domain. Tim4 tethers apoptotic cells through PS binding. Expression of the Tim4 transcript was unchanged in cKO rpMacs, whereas flow cytometric, Western and dot blot analyses revealed that Tim4 protein expression in cKO rpMacs was significantly lower than that in wild-type (WT) rpMacs. Moreover, the expression levels of other efferocytosis-related molecules, Mertk, Itgav and Itgb3, were normal in rpMacs. In addition, hypoglycosylated Tim4-FLAG fusion protein sufficiently recognized PS. These results demonstrated that core 1-derived O-glycan is required for Tim4-dependent normal efferocytosis and may contribute to the stable expression of the Tim4 glycoprotein.

Incomplete clearance of apoptotic cells by core 1-derived O-glycan-deficient resident peritoneal macrophages.

Chondroitin sulfate (CS) is a sulfated glycosaminoglycan (GAG) chain. In cartilage, CS plays important roles as the main component of the extracellular matrix (ECM), existing as side chains of the major cartilage proteoglycan, aggrecan. Six glycosyltransferases are known to coordinately synthesize the backbone structure of CS; however, their in vivo synthetic mechanism remains unknown. Previous studies have suggested that two glycosyltransferases, Csgalnact1 (t1) and Csgalnact2 (t2), are critical for initiation of CS synthesis in vitro. Indeed, t1 single knockout mice (t1 KO) exhibit slight dwarfism and a reduction in CS content in cartilage compared with wild-type (WT) mice. To reveal the synergetic roles of t1 and t2 in CS synthesis in vivo, we generated systemic single and double knockout (DKO) mice and cartilage-specific t1 and t2 double knockout (Col2-DKO) mice. DKO mice exhibited postnatal lethality, whereas t2 KO mice showed normal size and skeletal development. Col2-DKO mice survived to adulthood and showed severe dwarfism compared with t1 KO mice. Histological analysis of epiphyseal cartilage from Col2-DKO mice revealed disrupted endochondral ossification, characterized by drastic GAG reduction in the ECM. Moreover, DKO cartilage had reduced chondrocyte proliferation and an increased number of apoptotic chondrocytes compared with WT cartilage. Conversely, primary chondrocyte cultures from Col2-DKO knee cartilage had the same proliferation rate as WT chondrocytes and low GAG expression levels, indicating that the chondrocytes themselves had an intact proliferative ability. Quantitative RT-PCR analysis of E18.5 cartilage showed that the expression levels of Col2a1 and Ptch1 transcripts tended to decrease in DKO compared with those in WT mice. The CS content in DKO cartilage was decreased compared with that in t1 KO cartilage but was not completely absent. These results suggest that aberrant ECM caused by CS reduction disrupted endochondral ossification. Overall, we propose that both t1 and t2 are necessary for CS synthesis and normal chondrocyte differentiation but are not sufficient for all CS synthesis in cartilage.

Postnatal lethality and chondrodysplasia in mice lacking both chondroitin sulfate N-acetylgalactosaminyltransferase-1 and -2.

The glomerular filtration barrier is composed of podocytes, glomerular basement membrane, and endothelial cells. Disruption of these structures causes several glomerular injuries, such as focal segmental glomerulosclerosis (FSGS). The surface of podocyte apical membranes is coated by negatively charged sialic acids on core 1-derived mucin-type O-glycans. Here, we aimed to investigate the physiological role of core 1-derived O-glycans in the podocytes using adult mice lacking podocyte-specific core 1-derived O-glycans (iPod-Cos). iPod-Cos mice exhibited early and transient proteinuria with foot process effacements and developed typical FSGS-like disease symptoms. To identify the key molecules responsible for the FSGS-like phenotype, we focused on podocalyxin and podoplanin, which possess mucin-type O-glycans. Expression and localization of podocalyxin did not change in iPod-Cos glomeruli. Besides, western blot analysis revealed significantly lower levels of intact podocalyxin in isolated glomeruli of iPod-Cos mice, and high levels of processed forms in iPod-Cos glomeruli, as compared to that in control glomeruli. Conversely, podoplanin mRNA, and protein levels were lower in iPod-Cos mice than in control mice. These results demonstrated that core 1-derived O-glycan on podocytes is required for normal glomerular filtration and may contribute to the stable expression of podocalyxin and podoplanin.

Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis.

The core 1 structure is the major constituent of mucin-type -glycans, which are added via glycosylation-a posttranslational modification present on membrane-bound and secretory proteins. Core 1 β1,3-galactosyltransferase (C1galt1), an enzyme that synthesizes the core 1 structure, requires Cosmc, a C1galt1-specific molecular chaperone, for its enzymatic activity. Since -knockout mice exhibit embryonic lethality, the biological role of core 1-derived -glycans in the adult stage is not fully understood. We generated ubiquitous and inducible CAGCre-ER/-knockout (iCAG-Cos) mice to investigate the physiological function of core 1-derived -glycans. The iCAG-Cos mice exhibited a global loss of core 1-derived -glycans, high mortality, and showed a drastic reduction in weights of the thymus, adipose tissue, and pancreas 10 days after  deletion. They also exhibited leukocytopenia, thrombocytopenia, severe acute pancreatitis, and atrophy of white and brown adipose tissue, as well as spontaneous gastric ulcers and severe renal dysfunction, which were considered the causes underlying the high mortality of the iCAG-Cos mice. Serological analysis indicated the iCAG-Cos mice have lower blood glucose and total blood protein levels and higher triglyceride, high-density lipoprotein, and total cholesterol levels than the controls. These data demonstrate the importance of core 1-derived -glycans for homeostatic maintenance in adult mice.

Global Loss of Core 1-Derived -Glycans in Mice Leads to High Mortality Due to Acute Kidney Failure and Gastric Ulcers.

Sialic acid (SA) is present at the terminal ends of carbohydrate chains in glycoproteins and glycolipids and is involved in various biological phenomena. The biological function of the disialyl-T (SAα2-3Galβ1-3(SAα2-6)GalNAcα1-O-Ser/Thr) structure is largely unknown. To elucidate the role of disialyl-T structure and determine the key enzyme from the N-acetylgalactosaminide α2,6-sialyltransferase (St6galnac) family involved in its in vivo synthesis, we generated St6galnac3- and St6galnac4-deficient mice. Both single-knockout mice developed normally without any prominent phenotypic abnormalities. However, the St6galnac3::St6galnact4 double knockout (DKO) mice showed spontaneous hemorrhage of the lymph nodes (LN). To identify the cause of bleeding in the LN, we examined podoplanin, which modifies the disialyl-T structures. The protein expression of podoplanin in the LN of DKO mice was similar to that in wild-type mice. However, the reactivity of MALII lectin, which recognizes disialyl-T, in podoplanin immunoprecipitated from DKO LN was completely abolished. Moreover, the expression of vascular endothelial cadherin was reduced on the cell surface of high endothelial venule (HEV) in the LN, suggesting that hemorrhage was caused by the structural disruption of HEV. These results suggest that podoplanin possesses disialyl-T structure in mice LN and that both St6galnac3 and St6galnac4 are required for disialyl-T synthesis.

Sayaka Fuseya

Molecular & Cellular Glycoproteomics Research Group,
Cellular and Molecular Biotechnology Research Institute,
Molecular & Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute

National Institute of Advanced Industrial Science and Technology

Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System

Sayaka Fuseya

Molecular & Cellular Glycoproteomics Research Group,Cellular and Molecular Biotechnology Research Institute,Molecular & Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute

National Institute of Advanced Industrial Science and Technology

Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System

Molecular & Cellular Glycoproteomics Research Group,
Cellular and Molecular Biotechnology Research Institute,
Molecular & Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute