Characterization of Glycosaminoglycans by¹⁵N NMR Spectroscopy and in Vivo Isotopic Labeling

Abstract

Characterization of glycosaminoglycans (GAGs), including chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS), is important in developing an understanding of cellular function and in assuring quality of preparations destined for biomedical applications. While use of 1 H and 13 C NMR spectroscopy has become common in characterization of these materials, spectra are complex and difficult to interpret when a more heterogeneous GAG type or a mixture of several types is present. Herein a method based on 1 H-15 N two dimensional NMR experiments is described. The 15 N-and 1 H-chemical shifts of amide signals from 15 N-containing acetylgalactosamines in CSs are shown to be quite sensitive to the sites of sulfation (4-, 6-or 4,6-), and easily distinguishable from those of DS. The amide signals from residual 15 N-containing acetylglucosamines in HS are shown to be diagnostic of the presence of these GAG components as well. Most data were collected at natural abundance of 15 N despite its low percentage. However enrichment of the 15 N-content in GAGs using metabolic incorporation from 15 N-glutamine added to cell culture media is also demonstrated, and used to distinguish metabolic states in different cell types.

Unravelling Glycobiology by NMR Spectroscopy

“…In order to overcome respectively these two obstacles, methods of in vitro or in vivo isotopic labeling with specific magnetically active nuclear spins (principally 13 C and 15 N) [2,3] and new pulse sequences to specifically reduce losses from transverse relaxation mechanisms [4][5][6] were developed along the years. These methods, established primarily to proteins and nucleic acids [4][5][6][7][8][9], are now being used to study carbohydrates and glycoconjugates as well [10,11].…”

“…1 H-coupled 13 C-signals are very diagnostic of the methine (CH), methylene (CH2) and methyl (CH3) groups highly abundant in carbohydrate molecules, and thus quite useful in NMR structural glycobiology. In case of carbohydrates or glycosylated molecules that contain amino sugars, like the glycosaminoglycans (GAGs) that bear hexosamines with 15 N-natural abundant amino groups, the 15 N-related NMR signals although few in number are well-resolved and still quite useful for structural diagnosis [10]. Hence, hetero-atoms are very useful in solution NMR structural glycobiology through multi-dimensional heteronuclear experiments, or in direct observe experiments, as discussed next to describe the assignments of glucose and the novel method for characterizing GAG molecules through 15 N-atoms.…”

“…This is crucial, especially for those cases where the amount of material is a limitation, such as glycans isolated from cell cultures. Recently, we developed an in vivo method to spin-label cellular GAG molecules with nitrogen-15 [10] to allow observation and consequently get structural characterization via 15 N-chemical shift. This is the third isotope type used in NMR analysis in glycosaminoglycanomics.…”

“…This is the third isotope type used in NMR analysis in glycosaminoglycanomics. Our method was based on cell cultures using media enriched in 15 N-labeled side-chain glutamine [10]. The side-chain amino group of this amino acid is responsible to donate the NH2 group to the amino sugar during hexosamine biosynthesis in the cytosol.…”

“…The side-chain amino group of this amino acid is responsible to donate the NH2 group to the amino sugar during hexosamine biosynthesis in the cytosol. Through this in vivo labeling method, 15 N-HSQC spectra became recordable for a couple of few hundred micrograms of GAG isolated from cellular sources [10]. On the other hand, as standard GAGs can be readily available from suppliers, the HSQC spectra using 15 N-isotope at natural abundance proved to be successfully recordable at 15 mg/ml concentration-samples within just a couple of hours of acquisition.…”