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Csányi Mária Csilla

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Csányi Mária Csilla
Nanosurgical manipulation of extended von Willebrand factor multimers

Aug 29 - kedd

15:30 – 17:00

I. Poszterszekció

P07

Nanosurgical manipulation of extended von Willebrand factor multimers

Mária Csilla Csányi¹, Dominik Sziklai¹, Tímea Feller², Jolán Hársfalvi¹ and Miklós S.Z. Kellermayer¹

¹ Semmelweis University, Faculty of Medicine, Department of Biophysics and Radiation Biology

² University of Leeds, Leeds Institute of Cardiovascular and Metabolic Medicine, Discovery and Translational Science Department

The von Willebrand factor (VWF) is a varying-length multimeric chain of glycoprotein dimers/protomers with distinct domain structures. Conformational response of domains upon exposure to shear- and elongational forces ensures that VWF is able to mediate platelet adhesion to and aggregation on an injured vessel wall where it is explored or immobilized from the circulation. Human plasma-derived VWF multimers stretched by molecular combing extend through a specific hierarchy of structural intermediates of protomers as we have recently shown by using atomic force microscopy (AFM) [1]. However, the full scope of local domain stabilities and extensibilities remained hidden.

To uncover these cryptic structural details, in the present work, we used an in situ nanosurgical approach to probe whether targeted domains of VWF could be further extended and unfolded. To achieve this, surface-stabilized and pre-extended VWF multimers were manipulated at distinct spatial locations with the tip of the AFM cantilever. By moving the AFM tip in a direction perpendicular to the longitudinal axis of the VWF multimer, protein loops could be pulled out of the chain, the local extension of which was assessed following the acquisition of a subsequent topographic AFM image.

The extension resulted in ruptured and non-ruptured protein loops, with and without the appearance of hairpin-like thin sections and nodules, while the adjacent domains were non-displaced. Ruptures occurred in 27% of the VWF. Extension, which is the ratio of the length of the section following and prior to manipulation was 2.7 vs. 1.6, p= 0.0001 in non-ruptured and ruptured multimers. Nanomanipulation of protomers in which all the 5 nodules are separated, resulted in a mean final length of 345±69 nm, compared to their pre-manipulation length of 168±54 nm. None of the above results correlated with the protomer’s structural hierarchy showing the main role of the adhesion of certain domains to the mica surface. The nanosurgical manipulation used here demonstrates the VWF mechanical properties at the single domain level via extending further the C1-C6 and A1-A2-A3 domains or eventually rupturing them. The observed conformational changes indicate that VWF may have a large conformational force response potential in order to fine-tune the opening up of hidden epitopes for the different functions of the VWF.

Acknowledgment

TKP2021-EGA-23

References

[1] Csányi MC, Salamon P, Feller T, Bozó T, Hársfalvi J, Kellermayer MSZ. (2023) Protein Sci 32(1):e4535.

Sulea Cristina
Fibrillin-1 microfibrils in Marfan syndrome: nanoscale structural characterization using atomic force microscopy

Aug 29 - kedd

15:30 – 17:00

I. Poszterszekció

P27

Fibrillin-1 microfibrils in Marfan syndrome: nanoscale structural characterization using atomic force microscopy

Cristina M. Șulea^1,2,3, Zsolt Mártonfalvi¹, Csilla Csányi¹, Dóra Haluszka¹, Miklós Pólos^2,3, Kálmán Benke^2,3, Zoltán Szabolcs^2,3 and Miklós S. Z. Kellermayer¹

¹ Department of Biophysics and Radiation Biology, Semmelweis University, 1094 Budapest, Hungary

² Heart and Vascular Center, Semmelweis University, 1122 Budapest, Hungary

³ Hungarian Marfan Foundation, 1122 Budapest, Hungary

Fibrillin-1 microfibrils are essential elements of the extracellular matrix serving as a scaffold for the deposition of elastin and endowing connective tissues with tensile strength and elasticity. Mutations in the fibrillin-1 gene (FBN1) are linked to Marfan syndrome (MFS), a systemic connective tissue disorder that usually manifests in life-threatening aortic complications. The aortic involvement may be explained by a dysregulation in microfibrillar function and, conceivably, alterations in the microfibrils’ supramolecular structure.

The aim of the study was to perform a nanoscale structural characterization of fibrillin-1 microfibrils isolated from human aortic samples with different FBN1 gene mutations and to compare them with microfibrillar assemblies purified from non-MFS human aortic tissue.

Aortic wall samples were obtained from patients undergoing specific cardiovascular surgical interventions. Fibrillin-rich microfibrils were extracted by bacterial collagenase digestion and purified by size-exclusion chromatography. Atomic force microscopy was employed to visualize and study the microfibrillar assemblies.

Fibrillin-1 microfibrils displayed a characteristic “beads-on-a-string” appearance. The microfibrillar assemblies were investigated for bead geometry (height, length, and width), interbead region height, and periodicity. MFS fibrillin-1 microfibrils had a slightly higher mean bead height, but the bead length and width, as well as the interbead height, were significantly smaller in the MFS group. The mean periodicity varied around 50–52 nm among samples.

In conclusion, the data suggest an overall thinner and presumably more frail structure for the MFS fibrillin-1 microfibrils, which may play a role in the development of MFS-specific aortic symptomatology.

Acknowledgment

Funding sources: NRDI Office (ÚNKP-22-3-I-SE-49 to C.M.Ș.; K135360 to M.S.Z.K.; TKP2021-EGA-23), European Union (RRF-2.3.1-21-2022-00003 – National Cardiovascular Laboratory).