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Fibrillin secretion and microfibril assembly by Marfan dermal fibroblasts
Matrix Biology Vol. 14/1994, pp. 191 - 199 © 1994 by GustavFischerVerlag, Stuttgart. Jena • New York
Fibrillin Secretion and Microfibril Assembly by Marfan Dermal Fibroblasts CAY M. KIELTY 1, JANET E. PHILLIPS 1, ANNE H. CHILD 2, F. MICHAEL POPE 3 and C. ADRIAN SHUTTLEWORTH 1 1 School of BiologicalSciences,University of Manchester, Manchester, 2 St. George's Hospital Medical School, London and 3 MRC Clinical Research Centre, Northwick Park, UK.
Abstract The Marfan syndrome has been linked to the FBN1 gene encoding the microfibrillar glycoprotein fibrillin. To date, there have been no descriptions of microfibriUar abnormalities characteristic of this connective tissue disorder, although biochemical analyses have highlighted apparent abnormalities in fibrillin synthesis, secretion and processing. We have conducted a biochemical and ultrastructural investigation of fibrillin expression and assembly by a panel of dermal fibroblast lines from patients with Marfan syndrome and related diseases. The study has highlighted marked differences between cells in terms of secretion and aggregation of newly-synthesised fibrillin. In addition, electron microscopic visualization of fibrillin assemblies has clearly demonstrated for the first time the plethora of microfibrillar abnormalities that underlie this heterogeneous disorder. These data emphasize the molecular complexity that is a feature of the diverse clinical phenotypes exhibited by Marfan patients. Key words: Fibrillin, microfibril assembly, Marfan syndrome.
Introduction The glycoprotein fibrillin (Mr 350 000) has recently been identified as the principal structural component of a distinct class of extracellullar matrix microfibrils which are key determinants of connective tissue architecture and integrity (Sakai et al., 1986; 1991). Mutations in the FBN1 gene on chromosome 15, which encodes fibrillin, are the primary lesions in the Marfan syndrome, a heritable connective tissue disorder characterized by pleiotropic cardiovascular, skeletal and ocular abnormalities (Pyeritz, 1993). The clinically related disorders ectopia lentis and congenital contractural arachnodactyly have been linked to the FBN1 gene and a second fibrillin gene locus FBN2 on chromosome 5 respectively (Tsipouras et al., 1992). Rotary shadowing electron microscopy has revealed the complex architecture of the fibrillin-containing microfibrils isolated from tissues (Fleischmajer et al., 1991; Keene et al., 1991 a; 1991 b; Kielty et al., 1991; 1993) and cells (Kielty et
al., 1993). These macromolecules characteristically exhibit a diameter of 1 0 - 1 4 nm, a distinctive beaded morphology and an average, but variable, periodicity of 5 0 - 5 5 nm. However, the molecular interactions involved in fibrillin polymerisation and the macromolecular organization of assembled microfibrils remain to be defined. Fibrillin has a multi-domain structure with multiple epidermal growth factor (EGF)-like motifs interspersed with 8-cysteine repeats with homology to the TGF-[51 binding protein and several apparently unique cysteine-rich motifs (Maslen et al., 1991; Corson et al., 1993; Pereira et al., 1993). Calcium binding by EGF-like motifs and multiple intra- and intermolecular disulphide bonds are predicted to play a key role in stabilizing individual domains, fibrillin monomers and microfibrillar assemblies. To date, linkage and mutation analyses have been used to confirm clinical diagnoses of Marfan syndrome, and a number of FBN1 mutations have been identified in Marfan patients (Lee et al., 1991; Dietz et al., 1991; 1992a, b;
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1993; Kainulainen et al., 1991; 1992, 1993; Godfrey et al., 1993). There are a number of examples of missense mutations causing amino acid substitutions affecting cysteine or calcium-binding consensus residues within EGF-like domains, and nonsense mutations causing premature terminations and exon deletions. Efforts have also been made to develop diagnostic biochemical assays for fibrillin defects manifest at the protein level (McGookey-Milewicz et al., 1992; Aoyama et al., 1993). These studies have prompted the preliminary designation of patient categories which reflect abnormal fibrillin synthesis, secretion and/or deposition. At present, despite the progress that has been made in defining Marfan syndrome at gene and protein levels, no clear genotype-phenotype relationships have emerged and virtually nothing is known about how fibrillin mutations affect microfibril assembly and function. We have attempted to bridge this gap in our understanding of the processes spanning fibrillin mRNA translation and microfibril formation by conducting an extensive examination of fibrillin expression and assembly in a panel of Marfan dermal fibroblast lines. Biochemical approaches highlighted variations not only in fibrillin secretion and deposition, but also in the process of aggregation. Complementary ultrastructural examination of fibrillin assemblies elaborated in culture has highlighted the plethora of microfibrillar abnormalities chracteristic of this clinically heterogeneous disorder.
Materials and Methods
Materials Bacterial collagenase (type 1A), phenylmethanesulphonyl fluoride (PMSF), N-ethy|maleimide (NEM), Tween 20 and prestained molecular weight markers were obtained from the Sigma Chemical Company, Poole, Dorset, UK. CNBr-activated Sepharose CL-4B, Sepharose CL-2B, protein A-Sepharose and molecular weight markers were supplied by Pharmacia-LKB, Milton Keynes, Bucks, UK. Tissue culture media and plastics were obtained from Gibco BRL, Paisley, Scotland, UK. The radioisotope [3sS]TranSlabel was obtained from ICN Biomedicals Ltd., High Wycombe, Bucks., UK. Dermal fibroblasts were established by explant from skin biopsies from patients attending clinics at St. George's Hospital (Table l). Patients with Marfan syndrome presented with cardiovascular, skeletal and/ or ocular symptoms. Marfanoid patients exhibited some symptoms characteristic of Marfan syndrome patients. The ectopia lentis patient had, in addition to dislocated lenses, minor skeletal abnormalities. The congenital contractural arachnodactyly patient had pronounced skeletal defects.
Cells and cell culture Normal and Marfan dermal fibroblasts were routinely maintained in Dulbecco's minimum essential medium supplemented with 10% fetal calf serum, penicillin (400 units/ ml), streptomycin (50 ~tg/ml) and glutamine (200 ~tg/ml). Confluent cells between passages 3 and 6 were labelled for 18h with [3SS]-TranSlabel in medium containing 0.5% fetal calf serum. Labelled medium was fractionated by addition of solid ( N H 4 ) 2 S O 4 to 30% saturation at 4 °C in the presence of 5 mM NEM, 2 mM PMSF and 10 mM EDTA. Cell layers were sequentially extracted in 0.05 M Tris HCI, pH 7.4 containing 0.4 M NaC1, 0.005 M EDTA and 1% (v/v) Nonidet P40 (NET buffer) and 0.05 M Tris HCI, pH7.4 containing 4.0M guanidine HC1. Extracts were clarified by centrifugation for 15 min at 7500 x g on a bench microfuge, and soluble supernatants retained for analysis. For microfibril extractions, cells were maintained at post-confluence for up to three weeks. Cell layers were washed in 0.05 M Tris HC1, pH 7.4 containing 0.4 M NaC1, then incubated for 3 h at 20 °C in 0.05 M Tris HCI, pH 7.4 containing 0.4 M NaC1, 0.005 M CaC12, 0.1 mg/ml bacterial collagenase (type 1 A), 2 mM PMSF and 5 mM NEM. Soluble extracts were clarified by centrifugation for 15 rain at 7500 x g on a bench microfuge prior to size fractionation by gel filtration chromatography.
Size fractination of fibrillin solubilized from cell-layer extracts Guanidine HCI extracts of metabolically-labelled cell layers were chromatographed on gel filtration columns (1.5 x 25 cm) of Sepharose CL-2B (Kielty et al., 1993). Columns were equilibrated and eluted at room temperature with 0.05 M Tris HCI, pH 7.4 containing 4.0 M guanidine HCI. In each run, 60 1-ml fractions were collected. Column runs were constantly monitored at 280 nm and appropriate fractions pooled. The elution position of newly synthesized fibrillin was determined by counting fibrillin immunoprecipitates from pooled fractions. Post-confluent cell-layer extracts solubilized by bacterial collagenase digestion were chromatographed directly without concentration under non-reducing, non-denaturing conditions on Sepharose CL-2B equilibrated and eluted at room temperature with 0.05 M Tris HC1, pH 7.4, containing 0.4 M NaC1. High-Mr material present in the excluded volume (V0) was retained for ultrastructural analysis.
Immunoprecipitation Immunoprecipitation was carried out as described by Kiehy et al. (1990). Briefly, 30% ( N H 4 ) 2 S O 4 precipitates of cell culture medium were taken up in i ml NET buffer. Celllayer, guanidine HC1 extracts were dialysed extensively
M i c r o f i b r i l l a r A b n o r m a l i t i e s of M a r f a n S y n d r o m e
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Ultrastructural analysis
a g a i n s t 0 . 0 5 M Tris HC1, p H 7 . 4 c o n t a i n i n g 0.4 M N a C 1 a n d 0 . 0 0 5 M E D T A a n d 1 % (v/v) N o n i d e t P 4 0 w a s a d d e d p r i o r to i m m u n o p r e c i p i t a t i o n . In v i e w o f t h e s i m i l a r e l e c t r o p h o r e t i c m o b i l i t i e s o n SDSP A G E o f f i b r o n e c t i n a n d fibrillin, f i b r o n e c t i n w a s r e m o v e d p r i o r to i m m u n o p r e c i p i t a t i o n o f fibrillin b y t w o s e q u e n t i a l i n c u b a t i o n s w i t h 1 0 0 btl o f a 1:1 (v/v) s o l u t i o n o f g e l a t i n e S e p h a r o s e . S a m p l e s w e r e t h e n i n c u b a t e d for l h a t 2 0 °C w i t h a 1 : 1 0 0 d i l u t i o n o f a p o l y c l o n a l a n t i s e r u m to fibrillin m i c r o f i b r i l s ( S h u t t l e w o r t h et al., 1 9 9 2 ; Kielty et al., 1 9 9 3 ; W a g g e t t et al., 1 9 9 3 ) , p r i o r to t h e a d d i t i o n o f 6 0 ~tl o f a 1:1 (v/v) s o l u t i o n o f p r o t e i n A - S e p h a r o s e in N E T buffer.
V o i d v o l u m e f r a c t i o n s o f cell-layer e x t r a c t s w e r e visu a l i z e d for t h e i r m i c r o f i b r i l c o n t e n t by r o t a r y s h a d o w i n g using a modification of the mica sandwich technique (Kielty et al., 1993). I m m u n o g o l d e l e c t r o n m i c r o s c o p y w a s c a r r i e d o u t as d e s c r i b e d b y W a g g e t t et al. (1993).
Results
Synthesis and secretion of fibrillin T w e l v e M a r f a n d e r m a l f i b r o b l a s t lines, t h r e e M a r f a n o i d lines, a c o n g e n i t a l c o n t r a c t u r a l a r a c h n o d a c t y l y line a n d a n e c t o p i a lentis line w e r e i n v e s t i g a t e d b i o c h e m i c a l l y a n d u l t r a s t r u c t u r a l l y . T h e cells w e r e d e r i v e d f r o m clinically d i a g n o s e d p a t i e n t s ( T a b l e I). M e t a b o l i c a l l y - l a b e l l e d fibrillin w a s i m m u n o p r e c i p i t a t e d f r o m m e d i u m a n d cell-layer f r a c t i o n s o f ten p a t i e n t lines a n d n o r m a l c o n t r o l s a f t e r 18 h o f labelling. De novo fibrillin s y n t h e s i s w a s e x p r e s s e d as t o t a l c o u n t s i n c o r p o r a t e d i n t o fibrillin ( T a b l e II). T h e m a j o r i t y of n e w l y - s y n t h e s i s e d fibrillin w a s a l w a y s p r e s e n t w i t h i n t h e cell layers, as previo u s l y o b s e r v e d w i t h a r a n g e of n o r m a l cells (Kielty et al., 1993). H o w e v e r , t h e r e w e r e d i f f e r e n c e s in the d i s t r i b u t i o n o f n e w l y - s y n t h e s i s e d fibrillin b e t w e e n m e d i u m a n d cell-
Electrophoresis Immunoprecipitates were analysed by discontinuous S D S - P A G E o n 6 % gels ( L a e m m l i , 1 9 7 0 ) u n d e r n o n - r e d u c ing c o n d i t i o n s a n d by f l u o r o g r a p h y . M o l e c u l a r w e i g h t s w e r e d e t e r m i n e d by r e f e r e n c e to b o t h p r e s t a i n e d s t a n d a r d s [fumarase (56000), pyruvate kinase (65000), frucose-6p h o s p h a t e k i n a s e ( 8 8 0 0 0 ) , [3-galactosidase ( 1 2 5 0 0 0 ) , ct2 macroglobulin (190000)] and unstained standards ]bovine s e r u m a l b u m i n (67 0 0 0 ) , l a c t a t e d e h y d r o g e n a s e ( 1 4 0 0 0 0 ) , catalase (232000), ferritin (440000), thyroglobulin (669000)].
Table I. Clinical details and major phenotypic features of patients.
patient
cardiovascular ard/ag ad mvp
eh
ar
+ + + + + + + + + +
+ + + + + + + + -
skeletal sc/pd
jh
ocular rd
el
other my
pn
striae
+ -
+ -
-
+ + + +
+~
+
-
+
Marfan MC PW KD CS SHO DBE DBU JS RM SD KT US
+ + + +
+ + + -
+
-
+ + + + +
+ -
+ + - ? + + + + -
-/-/+ +/-/-/+ +/-/ . +/+/+ +/+ +/+/-
+ . + + . + + + + +
. .
. .
.
. + + + .
. . + . + -
.
. .
. .
+ + + + .
. + + + -
.
.
.
Marfanoid KB EG ES
-
JG
.
AP
+
-
+ + +
+ + +
+ +
-/+/+/-
+ + +
.
+
+
+ .
+ + .
.
.
ectopia lentis .
.
.
.
+/-
-
+
contractural arachnodactyly -
+
+
+
+/-
+
.
.
.
.
.
Abbreviations: ard, aortic root dilatation; ag, aortic regurgitation; ad, aortic dissection; mvp, mitral valve prolapse; eh, excessive height; ar, arachnodactyly; sc, scoliosis; pd, pectus deformities; jh, joint hypermobility; el, ectopia lentis; rd, retinal detachment; my, myopia; pn, pneumothorax. ~ in brother
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l
1
KD
AP
2
3
SHO
4
JG
JS
DBE
5
6
7
KB
8
B MC
SIlO PW
KB
Fig. 1. Electrophoretic analysis of fibrillin immunoprecipitated from medium and cell-layer extracts of patient cultures. Cells were labelled with [3sS]TranSlabel for 18 h. Samples were analysed by SDS-PAGE on 6% gels under non-reducing conditions and by fluorography. The electrophoretic mobilities of molecular weight markers catalase (Mr 232 000) and c~2-macroglobulin (Mr 190000) are indicated. (A) Medium was fractionated with (NH4)2SO4 to 30% saturation in the presence of inhibitors prior to immunoprecipitation of fibrillin. Secreted fibrillin components (Mr 300000 and 330000) were identified in the medium of eight cell lines. The Mr 300000 was present as a singlet or a doublet. Track1, MC; track2, KD; track3, AP; track4, SHO; track5, JG; track6, JS; track 7, DBE; track 8, KB. (B) Fibrillin was immunoprecipitated from guanidine HCIn extracted cell layers of four cell lines as a component of Mr 300 000, higher-Mr aggregates and lower-Mr fibrillin-immunoreactive bands. Track 1, MC; track2, SHO; track3, PW; track4, KB. Lower-Mr bands (arrowheads) were also apparent in SHO and KB immunoprecipitates.
Table II. Distribution of counts incorporated into newly-synthesised fibrillin by Marfan cell lines.
cell line control CS JG ES SHO RM JS DBE AP KD DBU KT
1
2
3
4
layer compartments in the different patient lines. In one case (MC), fibrillin counts were barely detectable. The electrophoretic mobility of fibrillin secreted into the medium by eight patient cell lines was determined by electrophoretic analysis of fibrillin immunoprecipitates on 6% SDS-PAGE gels under non-reducing conditions (Fig. 1A).
% total incorporated distribution of fibrillin, % in counts in fibrillin medium cell layer 0.142 0. 104 0.182 0.105 0.136 0.168 0.199 0.259 0.185 0.103 0.119 0.420
36 37 11 13 27 18 13 46 16 32 35 13
64 63 89 87 73 82 87 54 84 68 65 87
Cells were labelled for 18 h with [3sSlTranSlabel. Fibrillin was immunoprecipitated from 30% (NH4)2SO4 precipitates of cellculture medium and from guanidine HCI cell-layer extracts. The amount and distribution of newly-synthesised fibrillin in normal fibroblast culture and different patient cultures was compared by determining the percentage of total immunoprecipitated counts in medium and cell-layer extracts. The figures represent a single labellingexperiment in each case, but similar results were obtained from duplicate experiments. Fibrillin was present as a major component of M~ 3 0 0 0 0 0 which resolved as a doublet or singlet. An additional higher-Mr, fibrillin-immunoreactive component (Mr 330 000) was always present. Similar electrophoretic sep-
Microfibrillar Abnormalities of Marfan Syndrome
A
195
B 00 -
0.40-~____i ~~~o~ 1 ~~_~~ O.D.280
cpm 150
0.20 -
~
" - -
Z -
-
-
(D
Elutionvolume
F1 F2 F3
F4
Fig. 2. Aggregation of newly-synthesised fibrillin in cell layers. Guanidine HC1 cell-layer extracts were size fractionated by gel filtration chromatography on an analytical Sepharose CL-2B column (see Methods). The eluted volume was pooled into fractions 1-4 as indicated prior to fibrillin immunoprecipitation. Total fibrillin counts immunoprecipitated from pooled fractions were determined. The OD2s0 traces for all the cell-layer extracts were comparable. (A) Chromatography profile of SHO cell-layer extract. (B) Histograms showing total immunoprecipitated fibrillin counts in pooled fractions 1-4 (F1-F4) of SHO and MC cell-layerextracts. Open bars, SHO; hatched bars, MC. arations of fibrillin have previously been reported for normal and Marfan cells (Kielty et al., 1993; McGookeyMilewicz et al., 1992). A more complex electrophoretic pattern was observed when fibrillin was immunoprecipitated from cell-layer extracts of four of these lines (Fig. 1 B). Fibrillin was present both as a M r 300000 component and as higher-Mr aggregates; in addition, lower-Mr immunoreactive bands were detected.
Size fractionation of cell-layer, guanidine HCI extracts Aggregation of newly-synthesized fibrillin within the cell-layer compartments of patient cell lines soluble in guanidine HC1 was investigated by size fractionation of metabolically-labelled fibrillin (Fig. 2). Gel filtrationchromatography elution profiles of cell-layer extracts from all lines were comparable (Fig. 2A). Immunoprecipitation of labelled fibrillin from pooled column fractions demonstrated that fibrillin was present in all pooled fractions (Fig. 2B). The elution profiles of fibrillin reflected its size distribution within each cell layer. This approach highlighted marked fibrillin aggregation in the majority of lines, similar to that previously reported for a range of normal cells (Kielty et al., 1993). However, there was little evidence
for aggregation of fibrillin within cell layers of MC and PW cells.
Ultrastructural analyses Rotary shadowing electron microscopy of high-Mr assemblies isolated from post-confluent cell layers demonstrated that normal cultures, thirteen of the Marfan/Marfanoid lines, the ectopia lentis cells (JG) and the congenital contractural arachnodactyly cells (AP) respectively had elaborated microfibrils (Fig. 3). In the majority of cases, these were abundant and extensive assemblies, but in two lines (DBE and DBU), only a few intact microfibrils were detected. No intact microfibrillar assemblies were identified in cell-layer extracts of the remaining two Marfan cell lines (MC and PW). Microfibrils elaborated by normal fibroblasts were comparable in morphology to those previously isolated from tissues (Kielty et al., 1991, 1993) (Fig. 3 A). In contrast, all microfibrillar assemblies isolated from patient cell layers exhibited marked structural abnormalities (Fig. 3B-J). Nine patient cell lines (JS, RM, SD, KT, US, DBU, KB, EG and ES) assembled microfibrils that were diffuse with poorly defined interbead domain yet retained normal beaded periodicity (Fig. 3 B, C). Four lines (SHO, KD, DBE
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Fig. 3. Electron micrographs after rotary shadowing of fibrillin-containingmicrofibrils isolated from post-confluent cell layers. Microfibrils were isolated from post-confluent cell layers of normal and patient dermal fibroblasts. Fibrillin assemblies were clearly recognized by their distinctive beaded appearance. Microfibrils elaborated by the patient cells were morphologically abnormal compared to those isolated from normal cultures. The microfibrils shown are representative of all fields examined. Bars = 100 nm. Microfibrils extracted from (A) normal fibroblasts, (B) KB, (C) ES, (D) SHO, (E) KD, (F) DBE, (G) CS, (H) JG, (I) JG, (J) AP.
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197
The identity of the microfibrillar assemblies was confirmed in four lines (SHO, KB, EG and DBE) using immunogold localization of fibrillin (Fig. 4). Microfibrils elaborated by patient and control cells were recognized by anti-fibrillin serum (Kielty et al., 1993). Microfibrils from SHO cells were markedly irregular in outline in comparison to those elaborated by normal cells (Fig. 4). Fibrillinimmunoreactive material was also identified in cell-layer extracts of one of the two Marfan lines (PW) in which no assembled microfibrils were identified.
Discussion
Fig. 4. Electron micrographs after rotary shadowing of fibrillin assemblies treated with antifibrillin serum and second antibodyimmunogold conjugate. Immunogold localization of fibrillin epitopes present on fibrillin assemblies isolated from normal fibroblasts (A) and fibroblasts from patient SHO (B) confirmed the identity of microfibrillar assemblies elaborated by normal and Marfan fibroblasts. The SHO microfibrils were markedly irregular in outline compared to those elaborated by normal cells. Bars = 200nm. and CS) elaborated microfibrils with "frayed" interbead domains (Fig. 3 D - G ) . The beaded periodicity of KD microfibrils was variable (Fig. 3 E). Microfibrils elaborated by the ectopia lentis patient cells (JG) exhibited highly variable periodicity (Fig. 3H, I). In some fields, these assemblies were contracted, whilst in others the periodicity was so extended that microfibrillar integrity appeared compromised. The congenital contractural arachnodactyly line (AP) assembled abundant microfibrils with clearly disorganized interbead domains (Fig. 3J). These microfibrillar morphologies were clearly abnormal, apparent in all fields and reproducible for each cell line.
The recent linkage of the FBN1 gene to the Marfan syndrome has engendered an intensive search for specific mutations as a first step to defining genotype-phenotype relationships (Dietz et al., 1991; 1992a, 1992b, 1993; Godfrey et al., 1993; Kainulainen et al., 1991, 1992, 1993). In most inherited diseases, however, phenotype predictions based on specific mutations have not proved feasible. In the case of FBN1, which codes for a secreted protein that associates to form complex extracellular microfibrils, the relationship between a given mutation, microfibrillar dysfunction and clinical phenotype may be particularly complex. Efforts have also been made to develop diagnostic biochemical assays for defects in fibrillin expression (McGookey-Milewicz et al., 1992; Aoyama et al., 1993; Raghunath et al., 1993). Despite this recent deluge of information at gene and protein levels, the etiology of Marfan syndrome and the molecular basis of its clinical heterogeneity have to date eluded molecular definition. This is largely because current approaches have failed to address the central question concerning the consequences of mutations in terms of the structure and function of assembled microfibrils. Studies have been described where attempts were made to classify Marfan cell lines in terms of defects in fibrillin secretion and deposition (McGookey-Milewicz et al., 1991; Aoyama et al., 1993; Raghunath et al., 1993). Those studies addressed the question of processing but not the aggregation of fibrillin. This investigation has highlighted differences between dermal fibroblast lines from Marfan syndrome and related disorders with respect both to formation of aggregates in medium and cell layers and to elaboration of a fibrillin-rich matrix. It is not clear how these differences between cell lines might he related to clinical phenotype. Newly synthesised fibrillin (Mr 300 000) resolved as a doublet or singlet in the different patient lines. The significance of this electrophoretic pattern remains to be established, but by analogy with other inherited connective tissue diseases it is feasible that some lines secrete normal fibrillin and shorter monomers encoded by alleles with deletion mutations or premature terminations. Alterna-
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tively, defective glycosylation may give rise to a lower-Mr species. However, there is no consensus at present on the occurrence of fibrillin processing or its role in microfibril assembly. The identity of the higher-Mr fibrillinimmunoreactive component is unknown, but it could represent an unprocessed precursor or an immunologically related fibrillin-like molecule (McGookey-Milewicz et al., 1992). The lower-Mr bands in fibrillin immunoprecipitates of cell-layer extracts may be processed fibrillin fragments or molecules non-covalently associated with fibrillin. Ultrastructural examination clearly indicated that mutant fibrillin monomers were secreted and that micro fibrils elaborated within cell layers appeared structurally abnormal. The majority of patient cell lines were clearly capable of assembling periodic microfibrils which exhibited a range of distinct morphologies which presumably reflect specific mutations. This is the first visual evidence for fibrillin aggregation and microfibrillar abnormalities characteristic of Marfan patient cells. In many cases, microfibrils with diffuse, poorly defined interbead domains were observed. A fascinating feature of these microfibrils is that despite there disrupted organization, the periodicity remains remarkably constant, which raises the question of the molecular basis of this defined beaded periodicity. In general, the major clinical symptoms of these patients involve skeletal but not cardiovascular dysfunction, suggesting that a class of mutations exists that may not compromise the role of microfibrils in elastic fiber formation and function. At the other end of the spectrum, those patients for whom it was not possible to demonstrate the presence of assembled microfibrils in culture commonly have major cardiovascular involvement. These observations suggest a potential relationship between microfibrillar abnormalities and clinical phenotype and imply that the more structurally abnormal the isolated microfibrils appear, the more pronounced the cardiovascular and ocular symptoms. The observations also indicate that in the majority of Marfan cell lines products of mutated alleles are indeed secreted and incorporated into fibrillin assemblies. There are a number of examples of single base changes in FBN1 that give rise to amino acid substitutions, particularly in EGF-like motifs (Dietz et al., 1991, 1992 a, 1992 b; Kainulainen et al., 1992, 1993). Some of these mutations fall within calcium-binding consensus sequences of EGFlike motifs (Handford et al., 1991) and are predicted to disrupt calcium-binding and m o n o m e r conformation. In other cases, shortened products of mutated alleles (deletions or premature terminations) may be unable to align correctly to form the intermolecular interactions required to ensure microfibrillar integrity. These are potential molecular explanations for some of the microfibrillar abnormalities observed in this study. Despite the number of mutations that have now been reported and the relationship that has been established between FBN1 and Marfan syndrome, no comprehensive
molecular explanation of microfibril structure and function arising from these data has been derived to date. Indeed, it remains a possibility that mutations in genes other than FBN1 encoding microfibrillar proteins, such as other fibrillin isoforms or microfibril-associated glycoprotein (MAGP), may also give rise to microfibrillar abnormalities and Marfanoid symptoms. The approach outlined in this study provides a means to test structure-function predictions and how different mutations give rise to the heterogeneous clinical manifestations of Marfan syndrome. The elucidation of genotype-phenotype relationships will also undoubtedly shed light on the complex process of microfibril assembly.
This work was supported by the Medical Researcb Council and the Wellcome Trust. Dr. Child wishes to thank British Heart Foundation, Arthritis and Rheumatism Council, G.N.S. Trust, Marfan Trust, Abbeydale Trust and British Scoliosis Research Foundation for their generous support.
References
Aoyama, T., Gasner, C., Franke, U. and Furthmayr, H.: Classifica tion of Marfan syndrome fibroblast strains by fibrillin biosynthesis phenotype. Abstract, Ninth Annual Conference of National Marfan Foundation, Portland, Oregon. August, 1993. Corson, G.M., Chalberg, S.C., Dietz, H.C., Charbonneau, N. L. and Sakai, L. Y.: Fibrillin binds calcium and is coded by cDNAs that reveal a multidomain structure and alternatively spliced exons at the 5' end. Genomics 17: 476-484, ! 993. Dietz, H.C., Cutting, G.R., Pyeritz, R.E., Maslen, C.L., Sakai, L.Y., Corson, G.M., Puffenberger, E.G., Hamosh, A., Nanthakumar, E.J., Curristin, S.M., Stetten, G., Meyers, D.A. and Francomano, C.A.: Marfan syndrome is caused by a recurrent de novo missense mutation in the fibrillin gene. Nature 3.52: 337-339, 1991. Dietz, H.C., Pyeritz, R.E., Puffenberger, E.G., Kendzior, R.J., Corson, G.M., Maslen, C.L., Sakai, L.Y. and Francomano, C.A.: Marfan pbenotype variability in a family segregating a missense mutation in the epidermal growth factor-like motif of the fibrillin gene. J. Clin. Invest. 89:1674-1680, 1992 a. Dietz, H. C, Savaiva,J. M., Pyeritz, R. E., Cutting, G. R. and Frailcomano, C.A.: Clustering of fibrillin (FBN1) missense mutations in Marfan syndrome patients at cysteine residues in EGFlike domains. Hum. Mutat. I : 366- 374, 1992 b. Dietz, H. C., Valle, D., Francomano, C. A., Kendzior, R.]., Pyeritz, R.E. and Cutting, G. R.: The skipping of constitutive exons in vivo induced by nonsense mutations. Science 2.59: 680-683, 1993. Fleischmajer, R., Perlish, J. S. and Farraggiani, "[.: Rotary shadowing of collagen monomers, oligomers and fibrils during tendon fibrillogenesis. J. Histochem. Cytochem. 3 9 : 5 1 - 5 8 , 1991. Godfrey, M.: Molecular heterogeneity: a clinical dilemma. Clinical heterogeneity: a molecular dilemma. Am. J. Hum. Genet. 53: 22-25, 1993. Godfrey, M., Vandemark, N., Wang, M., Vellnov, M., Wargowski, D., Droste, S. and Rao, V. H.: Prenatal diagnosis and a donor splice site mutation in fibrillin in a family with Marfan syndrome. Am. J. Hum. Genet. .53: 472-480, 1993.
Microfibrillar Abnormalities of M a r f a n Syndrome Handford, P.A., Mayhew, M., Baron, M., Winship, P.R., Campbell, I.D. and Brownlee, G.G.: Key residues involved in calcium-binding motifs in EGF-like domains. Nature 351: 164-167, 1991. Kainulainen, K., Steinmann, B., Collins, F., Dietz, H.C., Francomano, C. A., Child, A. H., Kilpatrick, M. W. and Peltonen, L.: Marfan syndrome: no evidence for heterogeneity in different populations and more precise mapping of the gene. Am. J. Hum. Genet. 49: 662-667, 1991. Kainulainen, K., Sakai, L.Y., Child, A.H., Pope, F.M., Puhakka, L., Ryhanen, L., Palotie, A., Kaitila, I. and Peltonen, L.- Two mutations in Marfan syndrome resulting in truncated fibrillin polypeptides. Proc. Natl. Acad. Sci. USA 89: 5917-5921, 1992. Kainulainen, K., Karttunen, L., Puhakka, L., Sakai, L.Y. and Peltonen, L.: Ten novel mutations of the FBN1 resulting in a wide variety of clinical phenotypes. Nat. Genet. (in press), 1993. Keene, D.R., Sakai, L. Y. and Burgeson, R.E.- Human bone contains type IlI collagen, type VI collagen and fibrillin: type III collagen is present on specific fibres that may mediate attachment of tendons, ligaments and periosteum to calcified bone matrix. J. Histochem. Cytochem. 39: 5 9 - 6 9 , 1991 a. Keene, D.R., Maddox, B.K., Kuo, H.-J., Sakai, L.Y. and Glanville, R. W.: Extraction of beaded structures and their identification as fibrillin-containing matrix microfibrils. J. Histochem. Cytochem. 39: 441-449, 1991 b. Kielty, C.M., Boot-Handford, R.P., Ayad, S., Shuttleworth, C. A. and Grant, M.E.: Molecular composition of type VI collagen. Biochem. J. 272: 787-795, 1990. Kielty, C.M., Cummings, C., Whittaker, S. P., Shuttleworth, C. A. and Grant, M.E.: Isolation and ultrastructural analysis of microfibrillar structures from bovine elastic tissues. J. Cell Sci. 99: 797-807, 1991. Kielty, C.M., Berry, L., Whittaker, S.P. and Shuttleworth, C. A.: Microfibrillar assemblies of foetal bovine skin. Developmental distribution and relative abundance of type VI collagen and fibrillin. Matrix 13: 103-112, 1993. Kielty, C. M. and Shuttleworth, C.A.: Synthesis and assembly of fibrillin by fibroblasts and smooth muscle cells. J. Cell Sci. 106: 167-173, 1993. Laemmli, U. K.: Cleavage of structural proteins during assembly of the head of bactiophage T4. Nature 227: 680-685, 1970. Lee, B., Godfrey, M., Vitale, E., Hori, H., Mattei, M.-G., Sarfarazi, M., Tsipouras, P., Ramirez, E. and Hollister, D.W.: Linkage of Marfan syndrome and a phenotypically related disorder to two different fibrillin genes. Nature 352: 330-334, 1991.
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Maslen, C. L., Corson, G. M., Maddox, B. K., Glanville, R. W. and Sakai, L.Y.: Partial sequence of a candidate gene for Marfan syndrome. Nature 352: 334-337, 1991. McGookey-Milewicz, D., Pyeritz, R. E., Crawford, E. S. and Byers, P.H.: Marfan syndrome: defective synthesis, secretion and extracellular matrix formation of fibrillin by cultured dermal fibroblasts. J. Clin. Invest. 89: 79-86, 1992. Pereira, L., D'Alessio, M., Ramirez, F., Lynch,J. R., Sykes, B., Pangilinan, T. and Bonadio, J.: Genomic organization of the coding sequence for fibrillin, the defective gene product of Marfan syndrome. Hum. Molec. Genet. 2: 961-968, 1993. Pyeritz, R. E.: The Marfan syndrome. In: Connective Tissue and its Heritable Disorders, ed. by Royce, P. and Steinmann, B. WileyLiss, New York, 1993, pp. 437-468. Raghunath, M., Superti-Furga, A., Godfrey, M. and Steinmann, B.- Decreased deposition of fibrillin and decorin in neonatal Marfan syndrome fibroblasts. Hum. Genet. 90: 511-515, 1993. Sakai, L.Y., Keene, D.R., Glanville, R.W. and Bachinger, H.-P.: Purification and partial characterisation of fibrillin, a cysteinerich structural component of connective tissues. J. Biol. Chem. 266: 14763-14770, 1991. Sakai, L. Y., Keene, D. R. and Engvall, E.: Fibrillin, a new 350-kD glycoprotein is a component of extracellular microfibrils. J. Cell Biol. 103: 2499-2509, 1986. Shuttleworth, C. A., Berry, L. and Kielty, C. M.: Microfibrillar elements in dental pulp: Presence of type VI collagen and fibrillincontaining microfibrils. Arch. Oral Biol. 37: 1079-1084, 1992. Tsipouras, P., Del Mastro, R., Sarfarazi, M., Lee, B., Vitale, E., Child, A.H., Godfrey, M., Devereux, R.B., Hewitt, D., Steinmann, B., Viljoen, D., Sykes, B.C., Kilpatrick, M. and Ramirez, F.: Linkage analysis demonstrates that Marfan syndrome, dominant ectopia lentis and congenital contractural arachnodactyly are linked to the fibrillin genes on chomosomes 15 and5. N. Engl. J. Med. 326: 905-909, 1992. Waggett, A. D., Kielty, C.M. and Shuttleworth, C.A.: Micofibrillar elements in the synovial joint: presence of typeVI collagen and fibrillin-containing microfibrils. Ann. Rheum. Dis. 52: 449-453, 1993. Cay M. Kielty, School of Biological Sciences, University of Manchester, Manchester M 13 9PT, UK.
Fibrillin Secretion and Microfibril Assembly by Marfan Dermal Fibroblasts CAY M. KIELTY 1, JANET E. PHILLIPS 1, ANNE H. CHILD 2, F. MICHAEL POPE 3 and C. ADRIAN SHUTTLEWORTH 1 1 School of BiologicalSciences,University of Manchester, Manchester, 2 St. George's Hospital Medical School, London and 3 MRC Clinical Research Centre, Northwick Park, UK.
Abstract The Marfan syndrome has been linked to the FBN1 gene encoding the microfibrillar glycoprotein fibrillin. To date, there have been no descriptions of microfibriUar abnormalities characteristic of this connective tissue disorder, although biochemical analyses have highlighted apparent abnormalities in fibrillin synthesis, secretion and processing. We have conducted a biochemical and ultrastructural investigation of fibrillin expression and assembly by a panel of dermal fibroblast lines from patients with Marfan syndrome and related diseases. The study has highlighted marked differences between cells in terms of secretion and aggregation of newly-synthesised fibrillin. In addition, electron microscopic visualization of fibrillin assemblies has clearly demonstrated for the first time the plethora of microfibrillar abnormalities that underlie this heterogeneous disorder. These data emphasize the molecular complexity that is a feature of the diverse clinical phenotypes exhibited by Marfan patients. Key words: Fibrillin, microfibril assembly, Marfan syndrome.
Introduction The glycoprotein fibrillin (Mr 350 000) has recently been identified as the principal structural component of a distinct class of extracellullar matrix microfibrils which are key determinants of connective tissue architecture and integrity (Sakai et al., 1986; 1991). Mutations in the FBN1 gene on chromosome 15, which encodes fibrillin, are the primary lesions in the Marfan syndrome, a heritable connective tissue disorder characterized by pleiotropic cardiovascular, skeletal and ocular abnormalities (Pyeritz, 1993). The clinically related disorders ectopia lentis and congenital contractural arachnodactyly have been linked to the FBN1 gene and a second fibrillin gene locus FBN2 on chromosome 5 respectively (Tsipouras et al., 1992). Rotary shadowing electron microscopy has revealed the complex architecture of the fibrillin-containing microfibrils isolated from tissues (Fleischmajer et al., 1991; Keene et al., 1991 a; 1991 b; Kielty et al., 1991; 1993) and cells (Kielty et
al., 1993). These macromolecules characteristically exhibit a diameter of 1 0 - 1 4 nm, a distinctive beaded morphology and an average, but variable, periodicity of 5 0 - 5 5 nm. However, the molecular interactions involved in fibrillin polymerisation and the macromolecular organization of assembled microfibrils remain to be defined. Fibrillin has a multi-domain structure with multiple epidermal growth factor (EGF)-like motifs interspersed with 8-cysteine repeats with homology to the TGF-[51 binding protein and several apparently unique cysteine-rich motifs (Maslen et al., 1991; Corson et al., 1993; Pereira et al., 1993). Calcium binding by EGF-like motifs and multiple intra- and intermolecular disulphide bonds are predicted to play a key role in stabilizing individual domains, fibrillin monomers and microfibrillar assemblies. To date, linkage and mutation analyses have been used to confirm clinical diagnoses of Marfan syndrome, and a number of FBN1 mutations have been identified in Marfan patients (Lee et al., 1991; Dietz et al., 1991; 1992a, b;
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1993; Kainulainen et al., 1991; 1992, 1993; Godfrey et al., 1993). There are a number of examples of missense mutations causing amino acid substitutions affecting cysteine or calcium-binding consensus residues within EGF-like domains, and nonsense mutations causing premature terminations and exon deletions. Efforts have also been made to develop diagnostic biochemical assays for fibrillin defects manifest at the protein level (McGookey-Milewicz et al., 1992; Aoyama et al., 1993). These studies have prompted the preliminary designation of patient categories which reflect abnormal fibrillin synthesis, secretion and/or deposition. At present, despite the progress that has been made in defining Marfan syndrome at gene and protein levels, no clear genotype-phenotype relationships have emerged and virtually nothing is known about how fibrillin mutations affect microfibril assembly and function. We have attempted to bridge this gap in our understanding of the processes spanning fibrillin mRNA translation and microfibril formation by conducting an extensive examination of fibrillin expression and assembly in a panel of Marfan dermal fibroblast lines. Biochemical approaches highlighted variations not only in fibrillin secretion and deposition, but also in the process of aggregation. Complementary ultrastructural examination of fibrillin assemblies elaborated in culture has highlighted the plethora of microfibrillar abnormalities chracteristic of this clinically heterogeneous disorder.
Materials and Methods
Materials Bacterial collagenase (type 1A), phenylmethanesulphonyl fluoride (PMSF), N-ethy|maleimide (NEM), Tween 20 and prestained molecular weight markers were obtained from the Sigma Chemical Company, Poole, Dorset, UK. CNBr-activated Sepharose CL-4B, Sepharose CL-2B, protein A-Sepharose and molecular weight markers were supplied by Pharmacia-LKB, Milton Keynes, Bucks, UK. Tissue culture media and plastics were obtained from Gibco BRL, Paisley, Scotland, UK. The radioisotope [3sS]TranSlabel was obtained from ICN Biomedicals Ltd., High Wycombe, Bucks., UK. Dermal fibroblasts were established by explant from skin biopsies from patients attending clinics at St. George's Hospital (Table l). Patients with Marfan syndrome presented with cardiovascular, skeletal and/ or ocular symptoms. Marfanoid patients exhibited some symptoms characteristic of Marfan syndrome patients. The ectopia lentis patient had, in addition to dislocated lenses, minor skeletal abnormalities. The congenital contractural arachnodactyly patient had pronounced skeletal defects.
Cells and cell culture Normal and Marfan dermal fibroblasts were routinely maintained in Dulbecco's minimum essential medium supplemented with 10% fetal calf serum, penicillin (400 units/ ml), streptomycin (50 ~tg/ml) and glutamine (200 ~tg/ml). Confluent cells between passages 3 and 6 were labelled for 18h with [3SS]-TranSlabel in medium containing 0.5% fetal calf serum. Labelled medium was fractionated by addition of solid ( N H 4 ) 2 S O 4 to 30% saturation at 4 °C in the presence of 5 mM NEM, 2 mM PMSF and 10 mM EDTA. Cell layers were sequentially extracted in 0.05 M Tris HCI, pH 7.4 containing 0.4 M NaC1, 0.005 M EDTA and 1% (v/v) Nonidet P40 (NET buffer) and 0.05 M Tris HCI, pH7.4 containing 4.0M guanidine HC1. Extracts were clarified by centrifugation for 15 min at 7500 x g on a bench microfuge, and soluble supernatants retained for analysis. For microfibril extractions, cells were maintained at post-confluence for up to three weeks. Cell layers were washed in 0.05 M Tris HC1, pH 7.4 containing 0.4 M NaC1, then incubated for 3 h at 20 °C in 0.05 M Tris HCI, pH 7.4 containing 0.4 M NaC1, 0.005 M CaC12, 0.1 mg/ml bacterial collagenase (type 1 A), 2 mM PMSF and 5 mM NEM. Soluble extracts were clarified by centrifugation for 15 rain at 7500 x g on a bench microfuge prior to size fractionation by gel filtration chromatography.
Size fractination of fibrillin solubilized from cell-layer extracts Guanidine HCI extracts of metabolically-labelled cell layers were chromatographed on gel filtration columns (1.5 x 25 cm) of Sepharose CL-2B (Kielty et al., 1993). Columns were equilibrated and eluted at room temperature with 0.05 M Tris HCI, pH 7.4 containing 4.0 M guanidine HCI. In each run, 60 1-ml fractions were collected. Column runs were constantly monitored at 280 nm and appropriate fractions pooled. The elution position of newly synthesized fibrillin was determined by counting fibrillin immunoprecipitates from pooled fractions. Post-confluent cell-layer extracts solubilized by bacterial collagenase digestion were chromatographed directly without concentration under non-reducing, non-denaturing conditions on Sepharose CL-2B equilibrated and eluted at room temperature with 0.05 M Tris HC1, pH 7.4, containing 0.4 M NaC1. High-Mr material present in the excluded volume (V0) was retained for ultrastructural analysis.
Immunoprecipitation Immunoprecipitation was carried out as described by Kiehy et al. (1990). Briefly, 30% ( N H 4 ) 2 S O 4 precipitates of cell culture medium were taken up in i ml NET buffer. Celllayer, guanidine HC1 extracts were dialysed extensively
M i c r o f i b r i l l a r A b n o r m a l i t i e s of M a r f a n S y n d r o m e
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Ultrastructural analysis
a g a i n s t 0 . 0 5 M Tris HC1, p H 7 . 4 c o n t a i n i n g 0.4 M N a C 1 a n d 0 . 0 0 5 M E D T A a n d 1 % (v/v) N o n i d e t P 4 0 w a s a d d e d p r i o r to i m m u n o p r e c i p i t a t i o n . In v i e w o f t h e s i m i l a r e l e c t r o p h o r e t i c m o b i l i t i e s o n SDSP A G E o f f i b r o n e c t i n a n d fibrillin, f i b r o n e c t i n w a s r e m o v e d p r i o r to i m m u n o p r e c i p i t a t i o n o f fibrillin b y t w o s e q u e n t i a l i n c u b a t i o n s w i t h 1 0 0 btl o f a 1:1 (v/v) s o l u t i o n o f g e l a t i n e S e p h a r o s e . S a m p l e s w e r e t h e n i n c u b a t e d for l h a t 2 0 °C w i t h a 1 : 1 0 0 d i l u t i o n o f a p o l y c l o n a l a n t i s e r u m to fibrillin m i c r o f i b r i l s ( S h u t t l e w o r t h et al., 1 9 9 2 ; Kielty et al., 1 9 9 3 ; W a g g e t t et al., 1 9 9 3 ) , p r i o r to t h e a d d i t i o n o f 6 0 ~tl o f a 1:1 (v/v) s o l u t i o n o f p r o t e i n A - S e p h a r o s e in N E T buffer.
V o i d v o l u m e f r a c t i o n s o f cell-layer e x t r a c t s w e r e visu a l i z e d for t h e i r m i c r o f i b r i l c o n t e n t by r o t a r y s h a d o w i n g using a modification of the mica sandwich technique (Kielty et al., 1993). I m m u n o g o l d e l e c t r o n m i c r o s c o p y w a s c a r r i e d o u t as d e s c r i b e d b y W a g g e t t et al. (1993).
Results
Synthesis and secretion of fibrillin T w e l v e M a r f a n d e r m a l f i b r o b l a s t lines, t h r e e M a r f a n o i d lines, a c o n g e n i t a l c o n t r a c t u r a l a r a c h n o d a c t y l y line a n d a n e c t o p i a lentis line w e r e i n v e s t i g a t e d b i o c h e m i c a l l y a n d u l t r a s t r u c t u r a l l y . T h e cells w e r e d e r i v e d f r o m clinically d i a g n o s e d p a t i e n t s ( T a b l e I). M e t a b o l i c a l l y - l a b e l l e d fibrillin w a s i m m u n o p r e c i p i t a t e d f r o m m e d i u m a n d cell-layer f r a c t i o n s o f ten p a t i e n t lines a n d n o r m a l c o n t r o l s a f t e r 18 h o f labelling. De novo fibrillin s y n t h e s i s w a s e x p r e s s e d as t o t a l c o u n t s i n c o r p o r a t e d i n t o fibrillin ( T a b l e II). T h e m a j o r i t y of n e w l y - s y n t h e s i s e d fibrillin w a s a l w a y s p r e s e n t w i t h i n t h e cell layers, as previo u s l y o b s e r v e d w i t h a r a n g e of n o r m a l cells (Kielty et al., 1993). H o w e v e r , t h e r e w e r e d i f f e r e n c e s in the d i s t r i b u t i o n o f n e w l y - s y n t h e s i s e d fibrillin b e t w e e n m e d i u m a n d cell-
Electrophoresis Immunoprecipitates were analysed by discontinuous S D S - P A G E o n 6 % gels ( L a e m m l i , 1 9 7 0 ) u n d e r n o n - r e d u c ing c o n d i t i o n s a n d by f l u o r o g r a p h y . M o l e c u l a r w e i g h t s w e r e d e t e r m i n e d by r e f e r e n c e to b o t h p r e s t a i n e d s t a n d a r d s [fumarase (56000), pyruvate kinase (65000), frucose-6p h o s p h a t e k i n a s e ( 8 8 0 0 0 ) , [3-galactosidase ( 1 2 5 0 0 0 ) , ct2 macroglobulin (190000)] and unstained standards ]bovine s e r u m a l b u m i n (67 0 0 0 ) , l a c t a t e d e h y d r o g e n a s e ( 1 4 0 0 0 0 ) , catalase (232000), ferritin (440000), thyroglobulin (669000)].
Table I. Clinical details and major phenotypic features of patients.
patient
cardiovascular ard/ag ad mvp
eh
ar
+ + + + + + + + + +
+ + + + + + + + -
skeletal sc/pd
jh
ocular rd
el
other my
pn
striae
+ -
+ -
-
+ + + +
+~
+
-
+
Marfan MC PW KD CS SHO DBE DBU JS RM SD KT US
+ + + +
+ + + -
+
-
+ + + + +
+ -
+ + - ? + + + + -
-/-/+ +/-/-/+ +/-/ . +/+/+ +/+ +/+/-
+ . + + . + + + + +
. .
. .
.
. + + + .
. . + . + -
.
. .
. .
+ + + + .
. + + + -
.
.
.
Marfanoid KB EG ES
-
JG
.
AP
+
-
+ + +
+ + +
+ +
-/+/+/-
+ + +
.
+
+
+ .
+ + .
.
.
ectopia lentis .
.
.
.
+/-
-
+
contractural arachnodactyly -
+
+
+
+/-
+
.
.
.
.
.
Abbreviations: ard, aortic root dilatation; ag, aortic regurgitation; ad, aortic dissection; mvp, mitral valve prolapse; eh, excessive height; ar, arachnodactyly; sc, scoliosis; pd, pectus deformities; jh, joint hypermobility; el, ectopia lentis; rd, retinal detachment; my, myopia; pn, pneumothorax. ~ in brother
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l
1
KD
AP
2
3
SHO
4
JG
JS
DBE
5
6
7
KB
8
B MC
SIlO PW
KB
Fig. 1. Electrophoretic analysis of fibrillin immunoprecipitated from medium and cell-layer extracts of patient cultures. Cells were labelled with [3sS]TranSlabel for 18 h. Samples were analysed by SDS-PAGE on 6% gels under non-reducing conditions and by fluorography. The electrophoretic mobilities of molecular weight markers catalase (Mr 232 000) and c~2-macroglobulin (Mr 190000) are indicated. (A) Medium was fractionated with (NH4)2SO4 to 30% saturation in the presence of inhibitors prior to immunoprecipitation of fibrillin. Secreted fibrillin components (Mr 300000 and 330000) were identified in the medium of eight cell lines. The Mr 300000 was present as a singlet or a doublet. Track1, MC; track2, KD; track3, AP; track4, SHO; track5, JG; track6, JS; track 7, DBE; track 8, KB. (B) Fibrillin was immunoprecipitated from guanidine HCIn extracted cell layers of four cell lines as a component of Mr 300 000, higher-Mr aggregates and lower-Mr fibrillin-immunoreactive bands. Track 1, MC; track2, SHO; track3, PW; track4, KB. Lower-Mr bands (arrowheads) were also apparent in SHO and KB immunoprecipitates.
Table II. Distribution of counts incorporated into newly-synthesised fibrillin by Marfan cell lines.
cell line control CS JG ES SHO RM JS DBE AP KD DBU KT
1
2
3
4
layer compartments in the different patient lines. In one case (MC), fibrillin counts were barely detectable. The electrophoretic mobility of fibrillin secreted into the medium by eight patient cell lines was determined by electrophoretic analysis of fibrillin immunoprecipitates on 6% SDS-PAGE gels under non-reducing conditions (Fig. 1A).
% total incorporated distribution of fibrillin, % in counts in fibrillin medium cell layer 0.142 0. 104 0.182 0.105 0.136 0.168 0.199 0.259 0.185 0.103 0.119 0.420
36 37 11 13 27 18 13 46 16 32 35 13
64 63 89 87 73 82 87 54 84 68 65 87
Cells were labelled for 18 h with [3sSlTranSlabel. Fibrillin was immunoprecipitated from 30% (NH4)2SO4 precipitates of cellculture medium and from guanidine HCI cell-layer extracts. The amount and distribution of newly-synthesised fibrillin in normal fibroblast culture and different patient cultures was compared by determining the percentage of total immunoprecipitated counts in medium and cell-layer extracts. The figures represent a single labellingexperiment in each case, but similar results were obtained from duplicate experiments. Fibrillin was present as a major component of M~ 3 0 0 0 0 0 which resolved as a doublet or singlet. An additional higher-Mr, fibrillin-immunoreactive component (Mr 330 000) was always present. Similar electrophoretic sep-
Microfibrillar Abnormalities of Marfan Syndrome
A
195
B 00 -
0.40-~____i ~~~o~ 1 ~~_~~ O.D.280
cpm 150
0.20 -
~
" - -
Z -
-
-
(D
Elutionvolume
F1 F2 F3
F4
Fig. 2. Aggregation of newly-synthesised fibrillin in cell layers. Guanidine HC1 cell-layer extracts were size fractionated by gel filtration chromatography on an analytical Sepharose CL-2B column (see Methods). The eluted volume was pooled into fractions 1-4 as indicated prior to fibrillin immunoprecipitation. Total fibrillin counts immunoprecipitated from pooled fractions were determined. The OD2s0 traces for all the cell-layer extracts were comparable. (A) Chromatography profile of SHO cell-layer extract. (B) Histograms showing total immunoprecipitated fibrillin counts in pooled fractions 1-4 (F1-F4) of SHO and MC cell-layerextracts. Open bars, SHO; hatched bars, MC. arations of fibrillin have previously been reported for normal and Marfan cells (Kielty et al., 1993; McGookeyMilewicz et al., 1992). A more complex electrophoretic pattern was observed when fibrillin was immunoprecipitated from cell-layer extracts of four of these lines (Fig. 1 B). Fibrillin was present both as a M r 300000 component and as higher-Mr aggregates; in addition, lower-Mr immunoreactive bands were detected.
Size fractionation of cell-layer, guanidine HCI extracts Aggregation of newly-synthesized fibrillin within the cell-layer compartments of patient cell lines soluble in guanidine HC1 was investigated by size fractionation of metabolically-labelled fibrillin (Fig. 2). Gel filtrationchromatography elution profiles of cell-layer extracts from all lines were comparable (Fig. 2A). Immunoprecipitation of labelled fibrillin from pooled column fractions demonstrated that fibrillin was present in all pooled fractions (Fig. 2B). The elution profiles of fibrillin reflected its size distribution within each cell layer. This approach highlighted marked fibrillin aggregation in the majority of lines, similar to that previously reported for a range of normal cells (Kielty et al., 1993). However, there was little evidence
for aggregation of fibrillin within cell layers of MC and PW cells.
Ultrastructural analyses Rotary shadowing electron microscopy of high-Mr assemblies isolated from post-confluent cell layers demonstrated that normal cultures, thirteen of the Marfan/Marfanoid lines, the ectopia lentis cells (JG) and the congenital contractural arachnodactyly cells (AP) respectively had elaborated microfibrils (Fig. 3). In the majority of cases, these were abundant and extensive assemblies, but in two lines (DBE and DBU), only a few intact microfibrils were detected. No intact microfibrillar assemblies were identified in cell-layer extracts of the remaining two Marfan cell lines (MC and PW). Microfibrils elaborated by normal fibroblasts were comparable in morphology to those previously isolated from tissues (Kielty et al., 1991, 1993) (Fig. 3 A). In contrast, all microfibrillar assemblies isolated from patient cell layers exhibited marked structural abnormalities (Fig. 3B-J). Nine patient cell lines (JS, RM, SD, KT, US, DBU, KB, EG and ES) assembled microfibrils that were diffuse with poorly defined interbead domain yet retained normal beaded periodicity (Fig. 3 B, C). Four lines (SHO, KD, DBE
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Fig. 3. Electron micrographs after rotary shadowing of fibrillin-containingmicrofibrils isolated from post-confluent cell layers. Microfibrils were isolated from post-confluent cell layers of normal and patient dermal fibroblasts. Fibrillin assemblies were clearly recognized by their distinctive beaded appearance. Microfibrils elaborated by the patient cells were morphologically abnormal compared to those isolated from normal cultures. The microfibrils shown are representative of all fields examined. Bars = 100 nm. Microfibrils extracted from (A) normal fibroblasts, (B) KB, (C) ES, (D) SHO, (E) KD, (F) DBE, (G) CS, (H) JG, (I) JG, (J) AP.
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The identity of the microfibrillar assemblies was confirmed in four lines (SHO, KB, EG and DBE) using immunogold localization of fibrillin (Fig. 4). Microfibrils elaborated by patient and control cells were recognized by anti-fibrillin serum (Kielty et al., 1993). Microfibrils from SHO cells were markedly irregular in outline in comparison to those elaborated by normal cells (Fig. 4). Fibrillinimmunoreactive material was also identified in cell-layer extracts of one of the two Marfan lines (PW) in which no assembled microfibrils were identified.
Discussion
Fig. 4. Electron micrographs after rotary shadowing of fibrillin assemblies treated with antifibrillin serum and second antibodyimmunogold conjugate. Immunogold localization of fibrillin epitopes present on fibrillin assemblies isolated from normal fibroblasts (A) and fibroblasts from patient SHO (B) confirmed the identity of microfibrillar assemblies elaborated by normal and Marfan fibroblasts. The SHO microfibrils were markedly irregular in outline compared to those elaborated by normal cells. Bars = 200nm. and CS) elaborated microfibrils with "frayed" interbead domains (Fig. 3 D - G ) . The beaded periodicity of KD microfibrils was variable (Fig. 3 E). Microfibrils elaborated by the ectopia lentis patient cells (JG) exhibited highly variable periodicity (Fig. 3H, I). In some fields, these assemblies were contracted, whilst in others the periodicity was so extended that microfibrillar integrity appeared compromised. The congenital contractural arachnodactyly line (AP) assembled abundant microfibrils with clearly disorganized interbead domains (Fig. 3J). These microfibrillar morphologies were clearly abnormal, apparent in all fields and reproducible for each cell line.
The recent linkage of the FBN1 gene to the Marfan syndrome has engendered an intensive search for specific mutations as a first step to defining genotype-phenotype relationships (Dietz et al., 1991; 1992a, 1992b, 1993; Godfrey et al., 1993; Kainulainen et al., 1991, 1992, 1993). In most inherited diseases, however, phenotype predictions based on specific mutations have not proved feasible. In the case of FBN1, which codes for a secreted protein that associates to form complex extracellular microfibrils, the relationship between a given mutation, microfibrillar dysfunction and clinical phenotype may be particularly complex. Efforts have also been made to develop diagnostic biochemical assays for defects in fibrillin expression (McGookey-Milewicz et al., 1992; Aoyama et al., 1993; Raghunath et al., 1993). Despite this recent deluge of information at gene and protein levels, the etiology of Marfan syndrome and the molecular basis of its clinical heterogeneity have to date eluded molecular definition. This is largely because current approaches have failed to address the central question concerning the consequences of mutations in terms of the structure and function of assembled microfibrils. Studies have been described where attempts were made to classify Marfan cell lines in terms of defects in fibrillin secretion and deposition (McGookey-Milewicz et al., 1991; Aoyama et al., 1993; Raghunath et al., 1993). Those studies addressed the question of processing but not the aggregation of fibrillin. This investigation has highlighted differences between dermal fibroblast lines from Marfan syndrome and related disorders with respect both to formation of aggregates in medium and cell layers and to elaboration of a fibrillin-rich matrix. It is not clear how these differences between cell lines might he related to clinical phenotype. Newly synthesised fibrillin (Mr 300 000) resolved as a doublet or singlet in the different patient lines. The significance of this electrophoretic pattern remains to be established, but by analogy with other inherited connective tissue diseases it is feasible that some lines secrete normal fibrillin and shorter monomers encoded by alleles with deletion mutations or premature terminations. Alterna-
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tively, defective glycosylation may give rise to a lower-Mr species. However, there is no consensus at present on the occurrence of fibrillin processing or its role in microfibril assembly. The identity of the higher-Mr fibrillinimmunoreactive component is unknown, but it could represent an unprocessed precursor or an immunologically related fibrillin-like molecule (McGookey-Milewicz et al., 1992). The lower-Mr bands in fibrillin immunoprecipitates of cell-layer extracts may be processed fibrillin fragments or molecules non-covalently associated with fibrillin. Ultrastructural examination clearly indicated that mutant fibrillin monomers were secreted and that micro fibrils elaborated within cell layers appeared structurally abnormal. The majority of patient cell lines were clearly capable of assembling periodic microfibrils which exhibited a range of distinct morphologies which presumably reflect specific mutations. This is the first visual evidence for fibrillin aggregation and microfibrillar abnormalities characteristic of Marfan patient cells. In many cases, microfibrils with diffuse, poorly defined interbead domains were observed. A fascinating feature of these microfibrils is that despite there disrupted organization, the periodicity remains remarkably constant, which raises the question of the molecular basis of this defined beaded periodicity. In general, the major clinical symptoms of these patients involve skeletal but not cardiovascular dysfunction, suggesting that a class of mutations exists that may not compromise the role of microfibrils in elastic fiber formation and function. At the other end of the spectrum, those patients for whom it was not possible to demonstrate the presence of assembled microfibrils in culture commonly have major cardiovascular involvement. These observations suggest a potential relationship between microfibrillar abnormalities and clinical phenotype and imply that the more structurally abnormal the isolated microfibrils appear, the more pronounced the cardiovascular and ocular symptoms. The observations also indicate that in the majority of Marfan cell lines products of mutated alleles are indeed secreted and incorporated into fibrillin assemblies. There are a number of examples of single base changes in FBN1 that give rise to amino acid substitutions, particularly in EGF-like motifs (Dietz et al., 1991, 1992 a, 1992 b; Kainulainen et al., 1992, 1993). Some of these mutations fall within calcium-binding consensus sequences of EGFlike motifs (Handford et al., 1991) and are predicted to disrupt calcium-binding and m o n o m e r conformation. In other cases, shortened products of mutated alleles (deletions or premature terminations) may be unable to align correctly to form the intermolecular interactions required to ensure microfibrillar integrity. These are potential molecular explanations for some of the microfibrillar abnormalities observed in this study. Despite the number of mutations that have now been reported and the relationship that has been established between FBN1 and Marfan syndrome, no comprehensive
molecular explanation of microfibril structure and function arising from these data has been derived to date. Indeed, it remains a possibility that mutations in genes other than FBN1 encoding microfibrillar proteins, such as other fibrillin isoforms or microfibril-associated glycoprotein (MAGP), may also give rise to microfibrillar abnormalities and Marfanoid symptoms. The approach outlined in this study provides a means to test structure-function predictions and how different mutations give rise to the heterogeneous clinical manifestations of Marfan syndrome. The elucidation of genotype-phenotype relationships will also undoubtedly shed light on the complex process of microfibril assembly.
This work was supported by the Medical Researcb Council and the Wellcome Trust. Dr. Child wishes to thank British Heart Foundation, Arthritis and Rheumatism Council, G.N.S. Trust, Marfan Trust, Abbeydale Trust and British Scoliosis Research Foundation for their generous support.
References
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