Thomas Vorup-Jensen Lab

Research

The Laboratory of Biophysical Immunology aims to bring new methodologies from biophysics into studies of the immune system, in particular the function of b2 (CD18) integrins and complement, including the complement receptors (CR) 3 and CR4. Our work often addresses the connection between molecular architecture and function. This work is greatly aided by our collaborations with Aarhus University-based researchers as well as many researchers abroad. A main focus of the Biophysical Immunology Laboratory is to investigate the interplay between innate and adaptive immunity with the hypothesis that a major role of innate immunity is to regulate or guide the adaptive immune response. This consideration includes scenarios where aberrant activation of the innate immune system triggers an autoimmune response involving components of the adaptive immune system.

Concerning the role of conformational regulation of protein function studies of such changes in soluble molecules are important. The dimensions of mannan-binding lectin (MBL) were known from electron microscopy and well-suited for investigations with instruments found in the nanoscience armoury. By use of small-angle X-ray scattering (SAXS) the dimensions and molecular structure of MBL in solution was determined. By use of atomic force microscopy (AFM) we compared the structural properties of the molecule in solution to those of the molecule applied to surfaces with or without ligand, respectively. From these comparisons we concluded that MBL is stretched upon ligand-binding suggesting a model for the conformational changes that regulates the activation of the mannan-binding associated serine proteases and subsequently the complement cascade (1). This study was, in fact, the first experimental enquiry into a question, which has been the subject of speculations and demonstrates the powers of AFM and SAXS for investigating ultrastructural changes in proteins. Recently completed studies continued this work by probing the connection between the oligomeric structure and binding kinetics of MBL using SAXS and surface plasmon resonance (SPR) biosensors (2).

Prompted by these findings the conformational regulation of complement activation was studied by use of nanoparticles (3). The conformational regulation of IgM-mediated complement activation was demonstrated by the use dextran-coated nanoparticles as target from complement deposition. Pedersen et al. reported that only particles with an appropriate size would activate the complement system in a way that correlated with the titer of IgM antibodies to dextran. Based on classical work on the conformational changes in IgM accompanying the deposition on bacterial flagella, Pedersen et al. suggested that the curvature of the nanoparticle surface was a determinant in regulating the conformation of the surface-bound IgM. The biological implications were investigated by the use of nanoparticles of peptidoglycan, either generated by enzymatic digestion of purified peptidoglycan or as found in a planctonic culture of Staphylococcus aureus. Similar to the findings using engineered nanoparticles the results suggested that only peptidoglycan fragments of in a certain size regimen was able to efficiently activating the complement system. From investigation of the peptidoglycan fragments found in the culture medium it appeared that S. aureus liberate fragments with a surface curvature that would permit strong complement activation through IgM binding. Such particles could potentially act as decoy targets this way protecting S. aureus from complement mediated attack. Taken together with the analyses on conformational changes in MBL these findings point an exciting perspective on the role protein ultrastructure in the nanoscience of complement activation as discussed in a recent review by Vorup-Jensen & Boesen (4).

With regard to CR3 a surprising finding concerning its ligand binding properties came from a study of the pharmacological mode-of-action of glatiramer acetate (GA or Copaxone™). Originally designed as a peptide mimetic of myelin basic protein (MBP), a possible autoantigen in multiple sclerosis (MS), recent findings indicate that GA may modulate or suppress the inflammatory processes in such diverse pathophysiological settings as Alzheimer’s disease, graft-versus-host disease, and graft rejection following organ transplantation. Based on data from more than ten years of application in the clinic it now seems certain that GA has a benefit in MS treatment although the outcome of treatment of patients with relapsing-remitting MS is a moderate 30%-reduction of the frequency of attacks. Nevertheless the pharmacology of GA is poorly understood. Our laboratory discovered that the GA peptides bound CR3. By use of synchrotron radiation circular dichroism (SRCD) spectroscopy, it was possible to demonstrate that the GA peptides in solution take a largely unfolded structure, which is critical for the binding by CR3. CR3 was also found to bind MBP and GA may inhibit the binding by CR3-expressing cells to MBP at a concentration similar to the pharmaceutical dosage. A comparison of GA peptide-like sequences with the primary structure of MBP revealed that the GA peptides only mimic certain regions of the MBP sequence. By use of SAXS and SRCD spectroscopy these regions were further demonstrated to be largely unfolded in aqueous environments such body fluids, e.g., blood and cerebrospinal fluid (5). This has prompted the suggestion that the pharmacological mode of action of GA involves inhibition of the interaction between CR3 and MBP. With the expression of CR3 on macrophages and DCs such inhibition is likely to prevent the uptake of MBP in manner that would lead to presentation on MHC class II molecules (5).

The focus on chronic inflammation disorders, including autoimmune diseases, was recently further strengthened by the discovery that b2 integrins are shed from the leukocyte cell surface during synovial inflammation. In work together with Aarhus University Hospital, soluble complexes of b2 integrins (referred to as sCD11/CD18) were found at elevated level in synovial fluid from rheumatoid and spondylo arthritis patients compared with non-inflamed controls (6). In experiments with leukocytes in vitro tumor necrosis factor (TNF)a was demonstrated to enhance shedding (6), possibly through the activation of matrix metalloproteinase (MMP)-9, which has been reported to cleave CD18. Both TNFa and MMP-9 are implicated in mechanisms contributing to the development of chronic inflammation disorders and autoimmune diseases.  Surprisingly, an analysis by use of gel permeation chromatography demonstrated that both normal human plasma and synovial fluid contained oligomers of CD11/CD18 (6). By comparison with recombinant sCD11a/CD18 and a recent structural characterization of CR4 (CD11c/CD18) these complexes were suggested to be formed of oligomers of CD11/CD18, i.e., 2´CD11/CD18 and 4´CD11/CD18 (6). Although these complexes are apparently present in low concentrations in plasma the oligomerization permits avidity in the interaction with ICAM-1-coated surfaces such that the resulting dissociation constant take a value of KD~10-15 M. This implies that even at normophysiological conditions the sCD11/CD18 complexes will compete with the cellular expressed integrins for binding ICAM-1, which was supported by cell adhesion experiments in vitro. Since the sCD11/CD18 oligomerization is critical for obtaining such binding strength this points the oligomerization as a potentially important parameter in understanding the role of these complexes in normal biology as well as in patients with inflammation disorders. Interestingly, the sCD11/CD18 oligomnerization in the synovial fluid from rheumatoide and spondylo arthritis patients was markedly different (6). Current work focuses on understanding the cellular biology of the CD11/CD18 shedding, the protein chemistry of the oligomerization, and wider clinical significance of the shedding.

Research interests

  • Immunology
  • Innate immunity
  • Biophysics
  • Chronic Inflammation disorders
  • Nanoscience and nanotechnology
  • Protein structure

Methodologies

  • Receptor shedding
  • Cell migration assays
  • Cell adhesion assays
  • Complement activation
  • Small-angle X-ray scattering
  • Circular dichroism spectroscopy
  • Surface plasmon resonance

Collaborators and centres

  • Interdisiplinary Nanoscience Center (iNANO)
  • The Lundbeck Foundation Nanomedicine Center for Individualized Management of Tissue Damage and Regeneration (LUNA)

Research group members

  • Si Yang Huang, MSc, PhD, postdoc
  • Carina Rosenberg, MSc, PhD student
  • Babak Jalilian, DVM, MSc, PhD student
  • Halldór Bjarki Einarsson, MD, PhD student
  • Eva Kläning, PhD student
  • Technician Bettina W. Grumsen
  • 2 Bachelor students
  • 2 Master students

 

 

Group leader

Thomas Vorup-Jensen

Professor MSO
M
H bygn. 1242, 334
P +4587167853
P +4521489781

Publications

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Revideret 20.09.2017