In situ characterization of advanced glycation end products (AGEs) in collagen and model extracellular matrix by solid state NMR

Article abstract of DOI:10.1039/c7cc06624d

Non-enzymatic glycation of extracellular matrix with (U-¹³C₅)-d-ribose-5-phosphate (R5P), enables in situ 2D ssNMR identification of many deleterious protein modifications and crosslinks, including previously unreported oxalamido and

Full text of DOI:10.1039/c7cc06624d

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In situ characterization of advanced glycation
end products (AGEs) in collagen and model
extracellular matrix by solid state NMR†
Cite this:DOI: 10.1039/c7cc06624d
Received 23rd August 2017,
Accepted 14th September 2017
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R. Li, R. Rajan, W. C. V. Wong, D. G. Reid,
M. J. Duer,* V. J. Somovilla,
‡
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N. Martinez-Saez,‡ G. J. L. Bernardes,
R. Hayward and C. M. Shanahan
DOI: 10.1039/c7cc06624d
rsc.li/chemcomm
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Non-enzymatic glycation of extracellular matrix with (U-
5
C )- of circulating sugars, is a major factor underlying pathologies of
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D-ribose-5-phosphate (R5P), enables in situ 2D ssNMR identification diabetes, as well as normal ageing.
of many deleterious protein modifications and crosslinks, including biomedical importance to continue the characterization of
previously unreported oxalamido and hemiaminal (CH –CH(OH)NHR) glycation processes leading to advanced glycation end products
It is thus of great
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substructures. Changes in charged residue proportions and distribution (AGEs) and their effects on macroscopic and microscopic tissue
may be as important as crosslinking in provoking and understanding properties, and initiate an understanding of the effects of
harmful tissue changes.
glycation on biomolecular structure at the atomic level.
The complexity of the glycation process is compounded by
Glycation refers to spontaneously occurring non-enzymatic its proceeding with a distinct lack of consistency under apparently
reactions between biogenic aldehydes and ketones, in particular consistent conditions, rendering pathways and structural con-
the uncyclized forms of sugars, and biomolecular nucleophiles, sequences of glycation extremely difficult to study systematically
importantly the amino and guanidino side chains of protein Lys in vivo. This complexity is exacerbated by the occurrence of
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and Arg. Glycation is initiated by Schiff base formation followed reactions which are hard to predict even with simple model
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by Amadori rearrangement and thereafter a cascade of diverse compounds, and the poorly understood catalytic influence of
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and much studied but still incompletely characterized so-called certain biomolecules and ions.
‘Maillard’’ reactions. These can lead to adduction of basic groups Collagen is a major protein component of all vertebrate
‘
by acidic sugar glycoxidative breakdown products and thus tissues and the principle constituent of the extracellular matrix
changes in protein structure, net charge and charge distribution, (ECM) of connective tissue of bone, blood vessels, and numerous
and a variety of unnatural cross links between nearby residues. other organs. Apart from the mechanical and structural roles of
The effects of glycation are generally deleterious and result in the different connective tissue collagen isoforms, they all perform
altered tissue mechanical properties such as stiffness and tensile vital cell adhesion, motility, and signalling, roles the importance of
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strength,
net charge and charge distribution, and protein which is being increasingly appreciated. The collagens consist of
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binding recognition sites, resulting in altered molecular and repeating triplet amino acid motifs –(Gly–X–Y)– in which X and Y
cellular recognition, and triggering of inflammatory processes are frequently Pro and Hyp respectively, imposing a unique triple
via the ‘‘receptor of advanced glycation end products’’, RAGE.
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helical secondary structure on the three polypeptide chains, which
Predictably glycation is more severe in hyperglycemic states such comprises the fundamental building block of collagenous tissue,
as diabetes, and affects slow turnover macromolecules, such as including vascular smooth muscle (VSM) ECM. Collagen I triple
collagen, particularly markedly. Indeed glycation of connective helices self-associate into larger scale fibrillary structures,
tissues, especially blood vessels constantly exposed to high levels and additionally undergo a variety of orderly enzymatic post
translational modifications leading to glycosylations and cross
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linking at specific residues.
Department of Chemistry, University of Cambridge, Lensfield Road,
Cambridge CB2 1EW, UK. E-mail: mjd13@cam.ac.uk; Fax: +44(0)1223-336362;
Tel: +44(0)1223-736394
We have developed high yield methods of producing biomimetic
collagenous VSM cell (VSMC) ECM reproducibly in vitro. This
enables NMR active nuclei to be introduced into specific amino
acids, and studying of their structural environment, dynamics, and
chemistry, using powerful 2D (and potentially multidimensional)
solid state NMR methods impossible with unlabelled materials.
This provides a cost effective, and ethical, alternative to in vivo
b
BHF Centre of Research Excellence, Cardiovascular Division,
King’s College London, London SE5 9NU, UK
†
‡
Electronic supplementary information (ESI) available. See DOI: 10.1039/c7cc06624d
Current address: Department of Chemical Biology and Drug Discovery, Utrecht
Institute for Pharmaceutical Sciences, Bijvoet Center for Biomolecular Research,
Utrecht University, Universiteitsweg 99, Utrecht, Netherlands.
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methods for producing sufficient material for NMR. Equally unreacted materials (Fig. S1a, ESI†), in particular the prominent
importantly it facilitates the incorporation of specific amino signal at ca. 70 ppm from the g-carbon atoms of Hyp, an
acids, and sugars, (and in some cases, unavoidably, their effective NMR marker of collagenous tissue. It demonstrates
labelled metabolites) into matrix proteins for NMR structural that collagen is the major component of the in vitro material,
studies. We have used fetal sheep osteoblast (FSOb) ECM (FSOb- with minor components of other integral ECM proteins, and
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ECM) enriched in U- C, N-labelled Gly and Pro, particularly lipid. Incubation with U- C
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-R5P results in the appearance of a
prevalent in collagen, to validate our use of in vitro material number of new glycation product signals, which are qualitatively
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against labelled native bone. By incorporating specific labelled quite similar for the two materials although the extent of production
amino acids into in vitro ECM it is possible to address detailed of each is in many cases rather different (Fig. S1b, ESI†). The
aspects of collagen and ECM molecular structure and how this generation of new glycation product signals is clearly shown by a
changes under glycation. Ultimately by glycating matrix labelled comparison of the spectra of the glycated materials with those of
with glycation target residues Lys, and Arg, with labelled sugar pure collagen, and of R5P, which are overlaid in Fig. S1c (ESI†). In
reagents, identification of covalent AGE structures in situ may be order to assign these NMR signals, we have used 2D single
possible by ssNMR using similar atomic proximity-sensitive quantum–double quantum (SQ–DQ) and proton driven spin
techniques to those described below, without recourse to potentially diffusion (PDSD) correlation techniques.
destructive degradative analyses.
A first step in this process requires the identification of collagen, and of VSMC ECM, reacted with U- C
those NMR signals which arise from labelled glycating agent, method depends on direct transfer mediated by the comparatively
Accordingly Fig. 2 compares 2D SQ–DQ spectra of pure
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-R5P. As the
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and which from labelled glycated protein. Accordingly this weak C– C dipolar interaction, cross peaks effectively imply that
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communication reports 2D NMR characterization of labelled the corresponding signals are from mutually bonded C atoms
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products arising from reacting unlabelled native pure collagen, and therefore must originate from U- C -R5P. These connecti-
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and unlabelled in vitro VSMC ECM, with U- C labelled D-ribose- vities are also probed by the PDSD experiment, which reveals longer
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-phosphate (U- C
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-R5P), an important intermediate in nucleic range proximities as well depending on the experimental spin
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acid and energy metabolism,
and a vigorous endogenous diffusion time. PDSD datasets, which essentially corroborate
the SQ–DQ data, for the two glycated materials are compared in
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glycating agent.
Materials and methods, including the synthesis of the Fig. S2 (ESI†). Assignments are shown in Table 1, based in part
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sodium salt of U- C -R5P from commercial U- C -ribose, are on published data for a number of common glycation products
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described in detail in ESI.†
(CEL, pentosinane, DOGDIC, and DOPDIC, glucosepane,
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Fig. 1 compares the 1D C CP-MAS spectra of pure collagen GODIC, MODIC, DOGDIC, GOLD, and MOLD, and CML )
and VSMC ECM before, and after, incubation with U- C -R5P. and strong cross peaks due to polyhydroxylated structures. A
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There are close similarities between the spectra of the two cross peak attributable to a putative 5-phospho-ribuloselysine,
analogous to the initial ribuloselysine Amadori rearrangement
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product of
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C -ribose, is not observed because the 5-phosphate
group precludes cyclization to a stable furanose structure so this
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intermediate progresses rapidly to more advanced products.
Clear cross peaks in SQ–DQ spectra of both glycated materials
indicate the formation of a labelled fragment with two mutually
bonded carbon atoms with near equal chemical shifts of
ca. 165 ppm which we assign as part of an oxalate-derived
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structure. A distinct SQ–DQ correlation in U- C -R5P glycated
collagen (not seen in VSMC ECM) between bonded carbons at
shifts of 22 ppm and 91 ppm is consistent with a hemiaminal
AGE substructure CH CH(OH)NHR resulting from e.g. reaction
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of acetaldehyde with Lys. In general the products of the reaction
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between collagen and U- C -R5P qualitatively reproduce those
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observed from reaction with U- C -ribose. Although many
AGE structures observed by NMR are common to both pure
collagen and in vitro ECM there are quantitative differences,
most likely due to differences in overall protein composition,
and the accessibility of reactive groups to the glycating agents.
The in vitro ECM probably more closely replicates an in vivo
scenario as it would exist in the vasculature of a hyperglycemic
patient for instance, in containing a variety of other integral
ECM proteins besides type I collagen.
Fig. 1 1D spectra of unlabelled collagen (a), and VSMC ECM (b) before
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(black), and after (red), incubation with U- C -R5P. The Hyp g-carbon signal
Glycation is usually quantified using the natural fluores-
unique to collagen is labelled g-O. The VSMC ECM material also shows
signals at ca. 30 ppm and ca. 130 ppm from cross-polarizing lipid (lip.).
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cence of specific AGEs, and antibody probes raised against
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resulting from glycation of an immunogenic protein, clearly
leaving potential gaps in the AGE detection armory. Besides this
many characterization approaches, for instance hyphenated
chromatography–mass spectrometry, rely on hydrolysis of insoluble
proteins such as collagen to constituent glycation-modified amino
acids under rather severe conditions such as high temperatures and
acidity; successful detection of certain AGEs thus clearly depends on
their stability under these conditions. While NMR is considerably
less sensitive than the above techniques it possesses the unique
advantage that it can be applied to native glycated material with
negligible pre-treatment and consequent possible decomposition,
while the use of non-perturbing isotope labelled glycating agents is
straightforward and greatly increases the atomic level information
content of resulting data. Moreover our approach is directly
applicable to other native biomaterials such as, importantly,
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bone,
and in vitro model ECM.
It is widely assumed that changes in the mechanical and
consequently biological properties of ECM are due mainly to
the introduction of AGE induced crosslinks. While signals
consistent with some cross linking structures (pentosinane,
DOGDIC, DOPDIC, MODIC, GODIC) are observed in our materials,
our data suggests that the most abundant AGEs formed are rather
single amino acid residue modifications (CML, CEL, N-acetyl
species), and nitrogen adducts of single ribosyl (phosphate) units.
Such modifications convert basic protein functional groups into
charge neutral (N-acetyl, N-sugar adducts) or negative (CML, CEL)
substituents, with likely profound consequences for collagen triple
helical structure, interfibril associations, hydration, and molecular
recognition processes. Our results suggest that non-crosslinking,
monovalent glycation products may be at least as important as
Fig. 2 SQ–DQ correlation spectra of collagen (a), and VSMC ECM (b),
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after incubation with U- C -R5P. Correlations between significant glycation
products are marked in red; correlations between carbon atoms with identical
or very similar shifts are indicated *(vicinal diols) and **(hemiacetals, AGE crosslinks in modifying ECM mechanical and molecular
hemiaminal functionalities). The diagonal line represents the SQ–DQ axis.
recognition properties.
Dr Jonathan Clark of the Babraham Research Institute for
many helpful discussions; funding from the U.K. MRC (for DGR,
others. Such methods obviously depend on the AGEs of RR, RH), Royal Society (URF) and FCT Portugal (iFCT) (both for
interest being fluorescent in the first place, and the antigenicity GJLB), and for PhD studentships the China Scholarship Council
of specific already-known AGE structures or non-specific structures and Cambridge Trust (for RL), and U.K. EPSRC (for VWCW).
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Table 1 Summary of the C– C SQ–DQ and PDSD (100 ms spin diffusion mixing time) connectivities in pure collagen, and VSMC ECM, reacted with
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C -R5P, and comparison with products previously identified in pure collagen reacted with (U- C )-ribose
Cross peak correlations
Signal 1/ppm Signal 2/ppm
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10
Assignment
VSMC
Yes
Not obsd.
Yes
Collagen
U-
5
C -ribose collagen
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1
2
2
2
3
3
5
5
5
6
7
6
8
1
7
7
1
4
2
7
7
0
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7
2–84
4
4–73
8–92
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CEL and/or MODIC
Norpronyl lysine
N-Acetyls
Yes
Yes
Yes
Not obsd.
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Not certain
Yes
Yes
Not obsd.
Yes
193
173
180
91
59
69
172
176
193
62–84
104
178
Ca. 90
165
N-Acetyls
Yes
CH
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–CH(OH)NHR?
Not obsd.
Not obsd.
Weak
Yes
V. wk.
Weak
Yes
Yes
Yes
Yes
Yes
Not obsd.
Yes
Not obsd.
Yes
Yes
Yes
Yes
Yes
Yes
Yes
DOPDIC/pentosinane
DOPDIC
CML
CEL
Norpronyl lysine
Vicinal di-ols
Hydroxylic-(hemi)acetal carbons
DOGDIC, DOPDIC, MODIC, GODIC
(Hemi)acetal/aminal
Oxalic acid skeleton?
Yes
Not obsd.
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