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.

Proteome and Glycoproteome Analyses Reveal Regulation of Protein Glycosylation Site-Specific Occupancy and Lysosomal Hydrolase Maturation by N-Glycan-Dependent ER-Quality Control.

N-Glycan-dependent endoplasmic reticulum quality control (ERQC) primarily mediates protein folding, which determines the fate of the polypeptide. Monoglucose residues on N-glycans determine whether the nascent N-glycosylated proteins enter into and escape from the calnexin (CANX)/calreticulin (CALR) cycle, which is a central system of the ERQC. To reveal the impact of ERQC on glycosylation and protein fate, we performed comprehensive quantitative proteomic and glycoproteomic analyses using cells defective in N-glycan-dependent ERQC. Deficiency of MOGS encoding the ER α-glucosidase I, CANX, or/and CALR broadly affected protein expression and glycosylation. Among the altered glycoproteins, the occupancy of oligomannosidic N-glycans was significantly affected. Besides the expected ER stress, proteins and glycoproteins involved in pathways for lysosome and viral infection are differentially changed in those deficient cells. We demonstrated that lysosomal hydrolases were not correctly modified with mannose-6-phosphates on the N-glycans and were directly secreted to the culture medium in N-glycan-dependent ERQC mutant cells. Overall, the CANX/CALR cycle promotes the correct folding of glycosylated peptides and influences the transport of lysosomal hydrolases.

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.