X-Ray Diffraction Detects D-Periodic Location of Native Collagen Crosslinks In Situ and Those Resulting from Non- Enzymatic Glycation

Abstract

Synchrotron based X-ray diffraction experiments can be highly effective in the study of mammalian connective tissues and related disease. It has been employed here to observe changes in the structure of Extra-Cellular Matrix (ECM), induced in an ex vivo tissue based model of the disease process underlying diabetes. Pathological changes to the structure and organization of the fibrillar collagens within the ECM, such as the formation of nonenzymatic crosslinks in diabetes and normal aging, have been shown to play an important role in the progression of such maladies. However, without direct, quantified and specific knowledge of where in the molecular packing these changes occur, development of therapeutic interventions has been impeded. In vivo, the result of non-enzymatic glycosylation i.e. glycation, is the formation of sugar-mediated crosslinks, aka advanced glycation end-products (AGEs), within the native D-periodic structure of type I collagen. The locations for the formation of these crosslinks have, until now, been inferred from indirect or comparatively low resolution data under conditions likely to induce experimental artifacts. We present here X-ray diffraction derived data, collected from whole hydrated and intact isomorphously derivatized tendons, that indicate the location of both native (existing) and AGE crosslinks in situ of D-periodic fibrillar collagen.

Time scale of glycation in collagen of bovine pericardium-derived bio-tissues

“…constant length of gap and overlap region along the collagen fibers for both ribose and glucose solutions (Giannini et al, 2021). The variations of the relative electron density reported in Table 1 are expected to be related to glycation processes (Bailey et al, 1995;Hudson et al, 2018;Hadley et al, 1998Hadley et al, , 2001Madhurapantula & Orgel, 2017) as we will discuss.…”

“…The electron density profile shown in Fig. 3 is named gl , being ascribed to glycation processes occurring near the arginine and lysine amino acids of the collagen structure (Gautieri et al, 2014;Madhurapantula & Orgel, 2017). A magnification of the staggered repetition unit is shown in Fig.…”

“…4 (bottom), which shows a 2D projection of the triple-helix collagen residue, the lysine/hydroxylysine (orange bullets) and arginine (green bullets) amino acids positions. This arrangement is obtained by projecting the linearized 3D triple-helix collagen structure (Madhurapantula & Orgel, 2017), reporting the residue positions as fractional coordinates of the d M -period. The whole staggered repetition unit contains about 1050 residues, fractioned in about 4.5 parts (average value between the 5 staggered nanofibril fractions of the overlapping region and 4 of the gap region); 1050 divided by 4.5 gives 233 residues, and 65.5 nm divided by 233 gives 0.28 nm, which measures the rise of the collagen helix per residue.…”

Higher density of ECM composition in pancreatic cancer correlates with reduced drug delivery

“…The diffracted rays then pass through a flight tube and the pattern is recorded onto a detector. Figure taken from Madhurapantula, et al 9 with permission from authors.…”

“…By analyzing these diffraction patterns, it is possible to gain information about its relative abundance in a measured pathlength. 9 Over expression of the Epidermal Growth Factor Receptor (EGFR) is another common feature in many PDAC cases and, given the FDA approval of the anti-EGFR monoclonal antibody cetuximab, this receptor is often a molecular target for many immunotherapy-based treatments in metastatic disease. Although cetuximab has seen high success in many cancer types, it has seen limited therapeutic effectiveness in treatment of metastatic PDAC.…”