MSACL 2024 Abstract

Background: Congenital disorders of glycosylation (CDG) are one of the largest groups of inborn errors of metabolism with more than 170 types identified, most of which are under-studied. Among all the CDGs, PMM2-CDG is the most common and affects more than 50% of the CDG patients in the country. Recently, significant progress has been made in developing novel therapies for PMM2-CDG, including aldolase reductase inhibitors and mannose-1-phosphate replacement therapies. It is hypothesized that the clinical improvement and developmental gains achieved in affected children on these therapies may be due to increased monosaccharide flux into N-linked glycoprotein biosynthesis. This study aims to investigate the metabolic flux of stable isotope labeled 15N2-glutamine and 13C6-glucose into the glycoproteins from cultured human fibroblasts or mouse tissues post the whole-body metabolic tracing using in-depth high-resolution mass spectrometry (HRMS) methods.

Method: Both normal and PMM2-CDG human skin fibroblast cells were cultured in DMEM and dialyzed FBS with glutamine replaced by 15N2-glutamine. Mouse tissues were obtained after being intravenously infused with 15N2-glutamine for 4 hours. N-glycans from cells or tissues were released by PNGase F digestion, N-tagged with a modified quinoline and extracted using a HILIC plate in preparation for N-glycan heavy isotopic tracing analysis by UPLC-ESI-QTOF (G2Si Waters) and UHPLC-Orbitrap (IQ-X Tribrid Thermo Fisher) systems.

Results: We initiated metabolic tracing with 13.5 mM 15N2-glutamine in cultured skin fibroblasts when the cells reach 90% confluency for 24, 48, and 72 hours. On average, more than 20% GlcNAc in N-glycans was replaced with 15N1GlcNAc after 24 hrs of 15N2-glutamine tracing and the total 15N flux further increases at 48 and 72 hours. Interestingly, the rate of metabolic flux from glutamine to N-glycan is slow in PMM2-CDG cells compared to two concurrent control lines in a glycan specific pattern. On average, the rate of glutamine flux into high mannose glycans, Man6-9GlcNAc2, in PMM2-CDG cells is 70-80% of the control mean. As the number of GlcNAc in the N-glycan increases in the complexed glycans, such as Fuc1Gal2Man3GlcNAc4, the rate of flux dropped to 20% of the control mean, which could be explained by a reduced metabolic flux into the hexosamine biosynthetic pathway in PMM2-CDG cells. Using HRMS technology, we are able to detect as low as 1% 15N incorporating into GlcNAc in Fuc1GlcNAc2Man3GlcNAc2 glycan. As we examined the liver N-glycans extracted from a mouse infused with 15N2-glutamine through tail vein for 4 hours, a significant amount of 15N was detected in the GlcNAc fragments of mouse N-glycans.

Conclusion: Our metabolic N-glycan flux analysis using HRMS method was able to identify less than 1% metabolic flux through 15N2-glutamine and we can quantify the amount of flux as well as the rate of flux in cultured cells when there is >1% heavy isotopic incorporation. Furthermore, we provide evidence here that the metabolic flux through hexosamine biosynthetic pathway is reduced in a PMM2-CDG cell line and detectable within 72 hours of tracing, building a paradigm to study mammalian metabolic flux into N-linked protein glycosylation both in vitro and in vivo.