Calcium Determines the Shape of Fibrillin

Reinhardt, Dieter P.; Mechling, Diane E.; Boswell, Bruce A.; Keene, Douglas R.; Sakai, Lynn Y.; Bächinger, Hans Peter

doi:10.1074/jbc.272.11.7368

Cited by 99 publications

(81 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This is consistent with the behaviour of other well characterised cbEGF modules, such as those from fibrillin [19]. Protection against trypsin digestion is particularly significant since the activity of this enzyme is enhanced by calcium [20].…”

Section: Calcium Protects Cbegf 7-9 From Proteolytic Digestionsupporting

confidence: 86%

See 1 more Smart Citation

Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4

Periz

Gill

Knott

et al. 2005

Molecular and Biochemical Parasitology

View full text Add to dashboard Cite

Microneme proteins are secreted from apicomplexan parasites during invasion of host cells and they play crucial roles in parasite–host cell adhesion. EtMIC4 is a 240 kDa transmembrane protein from Eimeria tenella that contains 31 tandemly arranged epidermal growth factor (EGF), like repeats within its extracellular domain. The majority of these repeats have calcium binding (cb) consensus sequences. Little is known about cbEGFs in apicomplexan parasites but their presence in microneme proteins suggests that they may contribute to parasite–host interactions. To investigate the potential role of cbEGFs we have expressed and correctly refolded a cbEGF triplet from EtMIC4 (cbEGF7–9) and demonstrated that this triplet binds calcium. Circular dichroism spectroscopic analysis of cbEGF7–9 demonstrates that the molecule undergoes a gradual change in conformation with increasing levels of calcium. In the presence of calcium, the triplet becomes resistant to proteolytic degradation by a variety of proteases, a characteristic feature of cbEGF repeats from higher eukaryotic proteins, such as fibrillin, suggesting that calcium binding induces the formation of a rigid conformation. Moreover, mass spectrometric mapping of the cleavage sites that are protected by calcium shows that these sites are located both close to and distant from the calcium binding sites, indicating that protection is not due to steric hindrance by calcium ions, but rather due to the overall conformation adopted by the triplet in the presence of calcium. Thus, the tandemly-arranged cbEGF repeats within EtMIC4 provide a mechanism whereby, in the calcium-rich extracellular environment, the molecule could adopt a protease-resistant, rigid structure that could favour its interaction with host cell ligands

show abstract

Section: Calcium Protects Cbegf 7-9 From Proteolytic Digestionsupporting

confidence: 86%

“…Measurements of cbEGFs in the presence of calcium from the solution structure of a cbEGF dimer [7] and from rotary shadowing electron microscopy measurements of purified fibrillin [20] agree that a single cbEGF extends ∼2.9 nm in length. In the absence of calcium, the molecule adopts a wider conformation that is ∼30% shorter.…”

Section: Discussionmentioning

confidence: 65%

Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4

Periz

Gill

Knott

et al. 2005

Molecular and Biochemical Parasitology

View full text Add to dashboard Cite

show abstract

“…23,36 Indeed, CD of mfp-2 in the far UV was almost identical to that of oxidized recombinant human EGF, 22 29,37 and can be disrupted by temperature or reduction of disulfides. SFA studies have independently shown that mfp-2 is Ca 2þ binding 7 and a partially Ca 2þ -bound form of mfp-2 was apparently used in the CD studies.…”

Section: Discussionmentioning

confidence: 92%

Three intrinsically unstructured mussel adhesive proteins, mfp‐1, mfp‐2, and mfp‐3: Analysis by circular dichroism

Hwang

Waite

2012

Protein Science

View full text Add to dashboard Cite

Mussel foot proteins (mfps) mediate fouling by the byssal holdfast and have been extensively investigated as models for versatile polymer-mediated underwater adhesion and coatings. However, insights into the structural properties of mfps have lagged far behind the nanomechanical advances, owing in part to the inability of these proteins to crystallize as well as their limited solubility. Here, solution secondary structures of mfp-1, mfp-2, and mfp-3, localized in the mussel byssal cuticle, adhesive plaque, and plaque-substratum interface, respectively, were investigated using circular dichroism. All three have significant extended coil solution structure, but two, mfp-1 and mfp-2, appear to have punctuated regions of structure separated by unstructured domains. Apart from its punctuated distribution, the structure in mfp-1 resembles other structural proteins such as collagen and plant cell-wall proteins with prominent polyproline II helical structure. As in collagen, PP II structure of mfp-1 is incrementally disrupted by increasing the temperature and by raising pH. However, no recognizable change in mfp-1's PP II structure was evident with the addition with Ca 21 and Fe 31 . In contrast, mfp-2 exhibits Ca 21 -and disulfidestabilized epidermal growth factor-like domains separated by unstructured sequence. Mfp-2 showed calcium-binding ability. Bound calcium in mfp-2 was not removed by chelation at pH 5.5, but it was released upon reduction of disulfide bonds. Mfp-3, in contrast, appears to consist largely of unstructured extended coils.

show abstract

“…mAb 78 was produced (as we have described for other mAbs) from a fusion of splenocytes immunized with a pepsin fragment called PF3 (28) and has been characterized as an antibody specific for fibrillin-1. 5 Polyclonal antibody 9543 has been demonstrated to be highly specific for fibrillin-1 (30). Recombinant fibrillin-1 polypeptides were produced using human embryonic kidney 293 cells, purified, and characterized as described previously (3).…”

Section: Methodsmentioning

confidence: 99%

“…The molecular composition of these structures is assumed to be complex. However, based upon immunolocalization of fibrillin to these structures (2,3) and the shape of fibrillin monomers (4,5), fibrillins are thought to be the major backbone components of these extracted microfibrils. Fibrillincontaining microfibrils are ubiquitous in the connective tissue space (6), providing architectural support as well as information essential for appropriate signaling during morphogenesis (7)(8)(9).…”

mentioning

confidence: 99%

Effects of Fibrillin-1 Degradation on Microfibril Ultrastructure

Kuo

Isogai

Keene

et al. 2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Current models of the elastic properties and structural organization of fibrillin-containing microfibrils are based primarily on microscopic analyses of microfibrils liberated from connective tissues after digestion with crude collagenase. Results presented here demonstrate that this digestion resulted in the cleavage of fibrillin-1 and loss of specific immunoreactive epitopes. The proline-rich region and regions near the second 8-cysteine domain in fibrillin-1 were easily cleaved by crude collagenase. Other sites that may also be cleaved during microfibril digestion and extraction were identified. In contrast to collagenase-digested microfibrils, guanidine-extracted microfibrils contained all fibrillin-1 epitopes recognized by available antibodies. The ultrastructure of guanidine-extracted microfibrils differed markedly from that of collagenase-digested microfibrils. Fibrillin-1 filaments splayed out, extending beyond the width of the periodic globular beads. Both guanidine-extracted and collagenase-digested microfibrils were subjected to extensive digestion by crude collagenase. Collagenase digestion of guanidine-extracted microfibrils removed the outer filaments, revealing a core structure. In contrast to microfibrils extracted from tissues, cell culture microfibrils could be digested into short units containing just a few beads. These data suggest that additional cross-links stabilize the long beaded microfibrils in tissues. Based on the microfibril morphologies observed after these experiments, on the crude collagenase cleavage sites identified in fibrillin-1, and on known antibody binding sites in fibrillin-1, a model is proposed in which fibrillin-1 molecules are staggered in microfibrils. This model further suggests that the N-terminal half of fibrillin-1 is asymmetrically exposed in the outer filaments, whereas the C-terminal half of fibrillin-1 is present in the interior of the microfibril.Microfibrils have been extracted from a variety of tissues and visualized by electron microscopy as distinctive beaded string structures (1, 2). The molecular composition of these structures is assumed to be complex. However, based upon immunolocalization of fibrillin to these structures (2, 3) and the shape of fibrillin monomers (4, 5), fibrillins are thought to be the major backbone components of these extracted microfibrils. Fibrillincontaining microfibrils are ubiquitous in the connective tissue space (6), providing architectural support as well as information essential for appropriate signaling during morphogenesis (7-9). In human disorders associated with fibrillins, the structural integrity of fibrillin microfibrils is required for the proper function of certain connective tissues (10). Therefore, determination of the organization of fibrillin molecules within the microfibril is important basic information.The organization of fibrillin molecules within microfibrils has been controversial. Both parallel (head-to-tail) (3, 4) and antiparallel (11) arrangements of fibrillin molecules within microfibrils have been...

show abstract

Calcium Determines the Shape of Fibrillin

Cited by 99 publications

References 47 publications

Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4

Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4

Three intrinsically unstructured mussel adhesive proteins, mfp‐1, mfp‐2, and mfp‐3: Analysis by circular dichroism

Effects of Fibrillin-1 Degradation on Microfibril Ultrastructure

Contact Info

Product

Resources

About