Advances in Structural Biology of Glycosaminoglycans by 15 N-NMR Spectroscopy Vitor H. Pomin Universidade Federal do Rio de Janeiro

1. INTRODUCTION: Glycoconjugates

1. INTRODUCTION: GAG structures

GAGs 1. INTRODUCTION: GAG functions

1. INTRODUCTION: 15 N-NMR characterization of glycosaminoglycans (GAGs)  NMR characterization of GAGs is largely based on 1 H- and/or 13 C-resonances and analysis by 15 N is quite rare. DISADVANTAGE OF 15 N-NMR (poorly sensitive):  very low natural abundance (0.37%)  very low magnetic susceptibility ( ΔE=γђB 0 modulated by the low negative gyromagnetic ratio value of 15 N) Hence, 15 N-isotope labeling techniques are considerably required, although some analyses (industrial sources) still work at natural abundance! Table of some nuclei properties important for NMR detection. NuclideSpinNatural abundance Gyromagnetic ratio γ [10 7 rad T -1 s -1 ] NMR Frequency (at 18.8 Tesla) Proton ( 1 H)½ (1) Carbon-12 ( 12 C) Carbon-13 ( 13 C)½ (1/3.976) Nitrogen-14 ( 14 N) (1/13.831) Nitrogen-15 ( 15 N)½ (1/9.861)

1. INTRODUCTION: 15 N-NMR spectroscopy – Advantages of INEPT (Insensitive Nuclei Enhanced by Polarization Transfer) 1) An initial INEPT pulse train transfers polarization from 1 H to X via 1 J XH. (INEPT block) 2) The antiphase heteronuclear magnetization evolves during the variable evolution t1 period under the effect of X chemical shift. 3) Heteronuclear 1 H-X couplings are refocused by applying a 180º 1 H pulse at the middle of this period (see 1 H-decoupled X evolution block). 4) A retro-INEPT pulse train converts X magnetization to in-phase 1 H magnetization (see reverse-INEPT block). 5) Proton acquisition is performed with X decoupling (see 1 H-acquisition with X- decoupling).

1. INTRODUCTION: 15 N-NMR spectroscopy for proteins

4S 6S 2. RESULTS: 15 N-HSQC spectra of native GAGs – at natural abundance! Galactosaminoglycans - GalNAcGlycosaminoglycan - GlcNAc 3) Amide 1 H-resonaces of GalNAc-containing GAGs are more upfield than GlcNAc-containing GAGs 2) Amide 15 N-resonaces of 4-sulfated GalNAc units are more upfield than 6-sulfated units 1) The standard GAGs show amide proton resonances in distinct regions of the 1 H- 15 N HSQC spectra upfield downfield

1. RESULTS: 15 N-HSQC spectra of standard N-acetyl hexosamines α- D -N-Acetylglucosamine β- D -N-Acetylglucosamine δ H = 8.09 ppm δ H = ppm α- D -N-Acetylgalactosamine β- D -N-Acetylgalactosamine Amide 1 H-resonaces of GalNAc residues are more upfield than GlcNAc residues. upfield downfield

10 min 30 min 1,5 h 2,5h 2 days GlcGlcNH 2 GlcNAcGlcNS αH1 βH1 αH1 HOD 90% 10% 85% 15% 65% 35% 53% 47% 33% 67% 88% 12% 83% 17% 70% 30% 65% 35% 60% 40% 75% 25% 67% 33% 40% 60% 54% 46% 52% 48% 91% 9% 91% 9% 86% 14% 86% 14% 81% 19% βH1 αH1 βH1 αH1 βH1 1. RESULTS: α/β -anomeric ratio of glucosyl standards

1. RESULTS: 15 N-HSQC spectra of standard N-acetyl hexosamines α- D -N-Acetylglucosamine β- D -N-Acetylglucosamine δ H = 8.09 ppm δ H = ppm 45% 55% α- D -N-Acetylgalactosamine β- D -N-Acetylgalactosamine 45% 55% β β α α β-anomerics are less populated than α-anomerics in amino sugars.

1. RESULTS: 15 N-HSQC spectra of CS disaccharides 4S 6S 40% β-ΔC4S 60% α-ΔC4S 40% β-ΔC6S 60% α-ΔC6S δ H = 8.24 ppm δ H = 8.16 ppm β β α α

4S 6S 4S 6S 1. RESULTS: Sequencing of CS oligosaccharides by 15 N-HSQC

1. RESULTS: 15 N- 1 H assignment of N-acetylated glucosamines in Hep/HS (A) CHO HS (B) UFH ↓[GlcUA](C) HS - ↑[GlcUA] (75%) GlcNAc-GlcUA (25%) GlcNAc-IdoUA (17.7%) GlcNAc-GlcUA (82.3%) GlcNAc-IdoUA GlcNAc-GlcUA 13 C-HSQC A1 NS,6X -(I 2S ) I1 2S -(A NS,6x ) A2 NAc I5 2S G1 XS A1 NAc 15 N-HSQC

1. How do the sulfonamide group of Hep look like? Sinais 1 H- 15 N de GlcNAc

1. RESULTS: direct detection of 15 N (DNP-Hypersense) from 15 N-Gln vs GlcNS 23 mM 19 mM H 2 NCHC CH 2 OH O CH 2 C 15 NH 2 O Enhance ~4000 in the signal-to-noise ratio δ N = ppm At 500 MHz, this signal of 15 N would take ~ 1 month for same acquisition 20 mM δ N =93.1 ppm

αOH1 αOH4 αOH3 + αOH6,6’ αNH αH1 αH2 αH4 αH3 αH6 + αH6’ + αH5 βH6 βH2 βH3 βH6’ βH1 βNH 1H 1D (10:20:70% D 2 O:Acetone:H 2 O at 3 o C, pH 4.5) 1. RESULTS: making assignments in exchangeable 1 H of GlcNS αH2 αH4 αH2 αH4 NH N-N-

βNH (16%) αNH (84%) αNH 15 N-HSQC (10:20:70% D 2 O:Acetone:H 2 O at 3 o C) Hard to observe NHSO 3 - in HS/Hep samples using those special conditions!! The amine groups of GlcNAc residues seem to be deprotonated in solution!! 1. RESULTS: assignment of exchangeable anomeric 1 H in GlcNS

1. CONCLUSIONS: 15 N-NMR spectroscopy of GAG standards 1) In spite of the low NMR sensitive of 15 N-isotope in GAG molecules, experiments using standards can still be performed even at natural abundance. 2) The GAG types can be easily distinguished based on their distinct positions of cross-peaks in 1 H/ 15 N-HSQC spectra. 3) Although 15 N-HSQC of GAGs display only few peaks, they are still quite useful for structural identification such as: hexosamine type (GalNAc vs. GlcNAc), anomeric distribution ( α-/ β-ratio), types of chemical groups (4- and/or 6-sulfation in GalGs; N-sulfation and/or N-acetylation in GAGs), uronic acid type and content (IdoA vs. GlcA). Until now, these 15 N-NMR analysis of GAGs were possible mainly because of the availability of large amount of material (industrial sources)! What about this type of 15 N-NMR analyses on cellular GAGs? Development of an in vivo 15 N-labeling technique for cellular GAGs 4) Due to rapid exchange rates of amide- 1 H of N-sulfation in aqueous solution, the implementation of special conditions (very low temperature, organic solvents and well-controlled pHs) is required to allow observation of this group.

CoA ATPADP D -glucose D -glucose 6- phosphate fructose 6- phosphate Glucosamine 6-phosphate Glutamine Glutamate N-AcetylGlucosamine 6-phosphate N-AcetylGlucosamine 1-phosphate UDP-N-AcetylGlucosamineUDP-N-AcetylGalactosamine UTP Acetyl-CoA HEXOKINASE PHOSPHOGLUCOSE ISOMERASE ACETYLTRANSFERASE PYROPHOSPHORYLASE EPIMERASE * * * * ** CYTOSOL GLUTAMINE FRUCTOSE 6-PHOSPHATE AMIDOTRANSFERASE 2. INTRODUCTION: Biosynthetic pathway for 15 N-labeled N-acetyl-hexosamines PPi PHOSPHOGLUCOMUTASE

* * CYTOSOL 2. INTRODUCTION: Trafficking of UDP-hexoses into GOLGI for GAG biosynthesis

2. INTRODUCTION: GAG biosynthesis in GOLGI apparatus GOLGI APPARATUS 1. chain initiation 2. chain elongation 3. chain modification transferases sulfotransferases

2. RESULTS: 15 N-gHSQC spectra of cellular 15 N-labeled GAGs incubated 24 h with 15 N-Gln Yield of ~ 600 μg

2. RESULTS: isotopomeric MS analysis of the endothelial pure ΔC4S dimers incubated 24h with 15 N-Gln real spectrum simulated spectrum Natural abundance 8% 15 N-incorporation 10% 15 N-incorporation

2. RESULTS: 15 N-HSQC spectra of endothelial cellular GAGs incubated 85 h with 15 N-Gln Total GAGs ( μg) ~285 ~700 ~88 ~150 Disaccharide analyses -85% CS: (49% C4S), (11% C6S), (25% nonS) -82% CS: (67% C4S), (10% C6S), (23% nonS) -83% CS: (56% C4S), (10% C6S), (34% nonS) -85% CS: (62% C4S), (14% C6S), (24% nonS) -15% HS: (60% GlcNAc) (40% GlcNS) -18% HS: (100% nonS) -17% HS: (100% nonS) -15% HS: (100% nonS) Distribution of 1 5 N-hexosamine -100% 15 N-GalNAc (C4S) -25% 15 N-GalNAc (C4S) - 44% 15 N-GalNAc (C4S) -100% 15 N-GlcNAc-GlcA (HS nonS) -75% 15 N-GlcNAc-GlcA (HS nonS) -18% 15 N-GalNAc (C6S) -38% 15 N-GlcNAc Total labeling of 15 N -GalNAc – 8% -GalNAc – 19% -GalNAc – 31% -GalNAc – 3% -GlcNAc – 7% -GlcNAc – 49% - GlcNAc – 25% -GlcNAc – 22%

2. CONCLUSIONS: 15 N-NMR spectroscopy of cellular GAGs 1) Cellular 15 N-GAGs indeed can be produced by an in vivo labeling strategy using 15 N-glutamine enriched cellular media. 2) The cell types (including knock-out versions) revealed different proportion of GAGs and 15 N-incorporation. 3) The levels of 15 N-labeling depends on time of incubation, but also considerably on the genetic type of the cells (NDST-1 ko vs. NDST-1,2 ko vs. wild type). 4) Selective labeling of certain GAG types is also possible in manipulated systems, such as the high levels of 15 N-HS in NDST-1,2 ko endothelial cells.

4. ACKNOWLEDGEMENTS: