New method allows researchers to explore how fibrillin is assembled
Researchers in the department have shed light on the early stages of assembly of fibrillin into microfibrils, a major component of the elastic tissue of animals.
Fibrillin microfibrils form beaded filament structures (blue) in the extracellular matrix of tissues such as in the elastic laminae of the aorta. Click photo to enlarge.
The research from Professor Penny Handford's lab, with lead author Dr Sacha Jensen, is published in PNAS (1).
The work provides an important step in understanding how the extracellular matrix assemblies of microfibrils are constructed and how mutant fibrillin associated with human disorders behaves.
Fibrillins are a group of large, disulphide-rich molecules dominated by calcium-binding EGF-like (cbEGF) domains. Fibrillin monomers are assembled into microfibrils at the cell surface and are quickly cross-linked into the extracellular matrix. Here they contribute to the elastic properties of tissues such as blood vessels.
Unlike collagen, the other major connective tissue protein, fibrillin cannot be assembled in vitro. This has hampered studies of microfibril assembly and its regulation, which would be valuable in understanding how mutations in the fibrillin gene cause a number of human disorders such as Marfan Syndrome (MFS).
Dr Jensen and colleagues wanted to develop a cellular system that would allow them to investigate microfibril assembly. Using information from previous structural studies of the N-terminal domains of fibrillin-1 (2), they engineered a GFP-tagged version of the protein, inserting GFP into a flexible, glycine-rich region where it would not affect the behaviour of the protein.
The structure of fibrillin-1 with the furin cleavage site of the C-terminal propeptide shown. Information obtained from the structure of the N terminus (FUN-EGF3) was used to design a GFP-tagged fibrillin-1 construct.
To assess the ability of the recombinant protein (GFP-Fbn) to incorporate into microfibrils, the group established a co-culture system of HEK293T cells, transiently transfected with the fusion construct, and human skin fibroblasts.
'The HEK cells act as a protein factory only,' explains Dr Jensen. 'They deliver the protein to the fibroblasts which, unlike HEK cells, are able to assemble fibrillin.'
Using this system, the group could detect GFP-Fbn incorporated into microfibrils in the extracellular matrix. Because the GFP-Fbn is only transiently expressed, the behaviour of the fibrillin construct and its requirements for processing and assembly can be explored.
The group first probed a possible regulatory role for the conserved C-terminal propeptide in fibrillin-1, suggested from another study, by mutating amino acids at the propeptide cleavage site. The resulting unprocessed fibrillin retained its propeptide, was secreted from the cell, but could not assemble into microfibrils – indicating that removal of propeptide is important for assembly.
Further analysis showed that the propeptide together with a series of cbEGF domains at the C terminus are essential for secretion of full-length fibrillin-1 from cells.
GFP-Fbn fusion construct incorporates into microfibrils produced by cultured fibroblasts (white arrows). A mutant that blocks furin cleavage of the C-terminal propeptide (RRAA) does not incorporate into microfibrils.
'We looked at propeptide-truncated versions of fibrillin-1 with deletions of the C-terminus and found that if the propeptide is removed completely, it stops the protein getting out of the cell,' explains Dr Jensen. 'Even when the propeptide is present, secretion requires the presence of C-terminal cbEGF domains.'
The researchers believe this indicates a critical interaction between the two regions that could possibly function by preventing premature microfibril assembly in the cell.
Dr Jensen then used the system to find out more about the possible role of the C-terminal region in microfibril assembly. 'We looked at C-terminal variants that are secreted and found that when they get into the matrix, they don't assemble properly,' says Dr Jensen. 'We see irregular fragmented structures in the matrix where there would normally be even fibril structures.'
In a series of further experiments looking at retained mutant fibrillin-1, the group found that this does not interfere with wild-type fibrillin in cells. They also found that MFS-causing mutations located within the propeptide result in intracellular retention, with the protein apparently caught in the endoplasmic reticulum - consistent with protein misfolding.
From their work, the researchers suggest a model for the role of the propeptide in regulating microfibril assembly. They suggest that, in the secretory pathway, the propeptide masks a critical assembly site at the C terminus. Following cleavage of the propeptide at the cell surface, the exposed C terminus multimerises, leading to microfibril assembly.
The group hopes to be able to use their newly established system more widely. 'As well as being able to express other pathway-associated mutants to see whether they can be secreted or assembled, the system also allows us to start to get at the mechanisms of assembly,' adds Professor Handford.
1. Jensen, SA, Aspinall, G and Handford, PA. C-terminal propeptide is required for fibrillin-1 secretion and blocks premature assembly through linkage to domains cbEGF41-43. (2014) PNAS published ahead of print June 30, 2014, doi:10.1073/pnas.1401697111
2. Yadin, DA, Robertson, IB, McNaught-Davis, J, Evans, P, Stoddart, D, Handford, PA, Jensen, SA and Redfield, C. Structure of the fibrillin-1 N-terminal domains suggests heparan sulphate regulates the early stages of microfibril assembly. (2013) Structure 21:1743-1756