Functional consequences of homocysteinylation of the elastic fiber proteins fibrillin-1 and tropoelastin

Article abstract of DOI:10.1074/jbc.M109.021246

Homocystinuria caused by cystathionine-β-synthase deficiency represents a severe form of homocysteinemias, which generally result in various degrees of elevated plasma homocysteine levels. Marfan syndrome is caused by mutations in fibrillin-1, which is one of the major constituents of connective tissue microfibrils. Despite the fundamentally different origins, both diseases share common clinical symptoms in the connective tissue such as long bone overgrowth, scoliosis, and ectopia lentis, whereas they differ in others. Fibrillin-1 contains ～13% cysteine residues and can be modified by homocysteine. We report here that homocysteinylation affects functional properties of fibrillin-1 and tropoelastin. We used recombinant fragments spanning the entire fibrillin-1 molecule to demonstrate that homocysteinylation, but not cysteinylation leads to abnormal self-interaction, which was attributed to a reduced amount of multimerization of the fibrillin-1 C terminus. The deposition of the fibrillin-1 network by human dermal fibroblasts was greatly reduced by homocysteine, but not by cysteine. Furthermore, homocysteinylation, but not cysteinylation of elastin-like polypeptides resulted in modified coacervation properties. In summary, the results provide new insights into pathogenetic mechanisms potentially involved in cystathionine-β-synthase-deficient homocystinuria.

Full text of DOI:10.1074/jbc.M109.021246

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 2, pp. 1188–1198, January 8, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Functional Consequences of Homocysteinylation of the
Elastic Fiber Proteins Fibrillin-1 and Tropoelastin*
Received for publication, May 14, 2009, and in revised form, September 29, 2009 Published, JBC Papers in Press, November 4, 2009, DOI 10.1074/jbc.M109.021246
Dirk Hubmacher‡, Judith T. Cirulis§, Ming Miao§, Fred W. Keeley§, and Dieter P. Reinhardt‡¶1
From the ‡Faculty of Medicine, Department of Anatomy and Cell Biology, and the ¶Faculty of Dentistry, Division of
Biomedical Sciences, McGill University, Montreal, Quebec H3A 2B2 and the §Research Institute, The Hospital for Sick Children,
Toronto, Ontario M5G 1X8, Canada
Homocystinuria caused by cystathionine-␤-synthase defi-
ciency represents a severe form of homocysteinemias, which
generally result in various degrees of elevated plasma homocys-
teine levels. Marfan syndrome is caused by mutations in fibril-
lin-1, which is one of the major constituents of connective tissue
microfibrils. Despite the fundamentally different origins, both
diseases share common clinical symptoms in the connective tis-
sue such as long bone overgrowth, scoliosis, and ectopia lentis,
whereas they differ in others. Fibrillin-1 contains ϳ13% cysteine
residues and can be modified by homocysteine. We report here
that homocysteinylation affects functional properties of fibril-
lin-1 and tropoelastin. We used recombinant fragments span-
ning the entire fibrillin-1 molecule to demonstrate that
homocysteinylation, but not cysteinylation leads to abnormal
self-interaction, which was attributed to a reduced amount of
multimerization of the fibrillin-1 C terminus. The deposition of
the fibrillin-1 network by human dermal fibroblasts was greatly
reduced by homocysteine, but not by cysteine. Furthermore,
homocysteinylation, but not cysteinylation of elastin-like
polypeptides resulted in modified coacervation properties. In
summary, the results provide new insights into pathogenetic
mechanisms potentially involved in cystathionine-␤-synthase-
deficient homocystinuria.
olism result in elevated blood levels of homocysteine, ranging
from 15 to 20 ␮M (mild forms) up to 500 ␮M (severe forms),
compared with 5–10 ␮M under normal conditions (2–4).
Fibrillins belong to a family of modular extracellular matrix
proteins including three isoforms, fibrillin-1, -2, and -3, and the
latent transforming growth factor-␤-binding proteins (for a
recent review, see Ref. 5). The most abundant domains in fibril-
lins are characterized by 6–8 intra-domain disulfide bonds and
include the calcium-binding epidermal growth factor-like
(cbEGF) domain and the transforming growth factor-␤ binding
protein-like domain. These domains represent individual fold-
ing units and are essential to maintain the structural integrity
and functional properties of fibrillins (for review, see Ref. 6).
The most common mutations reported to cause Marfan syn-
drome delete or generate cysteines in these domains, leading to
the presence of at least one unpaired cysteine residue (7).
Selected mutations of this group result in a modification of the
secondary structure in vitro, rendering the molecules suscepti-
ble to proteolytic degradation (8, 9).
The functional entities of fibrillins in tissues are high molec-
ular weight multiprotein assemblies, called microfibrils (10–
12). Disulfide bond-mediated multimerization of the fibrillin-1
C terminus and N- to C-terminal self-interaction are thought to
be the initial steps in the formation of tissue microfibrils (13–
15). Microfibrils were suggested to serve as a scaffold for the
deposition of tropoelastin and are important for the mainte-
nance of elastic fibers in skin, lung, and aorta (16). This hypoth-
esis has been further substantiated by the analysis of fibrillin-1
and -2 double null mice that show only traces of elastic fibers
between smooth muscle cells in the developing aorta versus
much more organized lamellar units visible in wild-type ani-
mals (17, 18). In addition to the role as a structural scaffold,
microfibrils serve as a reservoir for growth factors such as trans-
forming growth factor-␤ or bone morphogenetic proteins
(19–22).
Homocystinuria (OMIM ϩ236200) and Marfan syndrome
(OMIM #154700) share some common clinical symptoms such
as ectopia lentis, long bone overgrowth, and scoliosis, but differ
significantly in other symptoms. Marfan syndrome is caused by
mutations in fibrillin-1, whereas homocystinuria is caused in
most cases by cystathionine-␤-synthase (CBS)2 deficiency, an
enzyme that converts homocysteine to cystathionine resulting
in highly elevated homocysteine and methionine and reduced
cysteine concentrations (1). In addition, various defects or poly-
morphisms in genes for the methionine and vitamin B12 metab-
Elastic fibers are essential entities in skin, blood vessels, and
lung, where they confer tissue elasticity. During elastic fiber
biogenesis, monomeric tropoelastin forms small aggregates on
the cell surface, which are deposited on the microfibril scaffold.
After cross-linking and other maturation steps, they are trans-
formed into a highly insoluble and mechanically durable mate-
rial (18, 23). Tropoelastin contains two cysteine residues that
are located in the conserved C terminus, where they form an
intra-molecular disulfide bond (24). The self-assembly process
for tropoelastin and the properties of elastin-like polypeptides
can be experimentally assessed in a temperature-induced phase
* This work was supported by a grant from the Canadian Marfan Association
(to D. P. R.), Canadian Institutes of Health Research Grant MOP-68836 (to
D. P. R.), the German Academic Exchange Service (to D. H.), and Heart and
Stroke Foundation of Ontario Grant T5451 (to F. W. K.).
1 To whom correspondence should be addressed: 3640 University St., Mon-
treal, QC H3A 2B2, Canada. Tel.: 514-398-4243; Fax: 514-398-5375; E-mail:
dieter.reinhardt@mcgill.ca.
2 The abbreviations used are: CBS, cystathionine-␤-synthase; BSA, bovine
serum albumin; cbEGF, calcium-binding epidermal growth factor-like
domain; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol;
HSF, primary human skin fibroblasts; MES, 2-(N-morpholino)ethanesulfo-
nic acid; PBS, phosphate-buffered saline; TBS, Tris-buffered saline.
1188 JOURNAL OF BIOLOGICAL CHEMISTRY
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Homocysteinylation of Elastic Fiber Proteins
separation, known as coacervation (25, 26). This process is de- GTTTCCCGGCTTT-3Ј; Ex24/36-EcoRI, 5Ј-AGGGAAT-
pendent on factors such as ionic strength, pH, polypeptide con- TCCTATTTTCTCTTCCGGCCACAAGCTTTCCCCAGGC-
centration, and the presence of other extracellular matrix pro- AGGCCCCACCAGGGCCAATCGCGGGA-3Ј.
teins (27–31).
The corresponding PCR product (ϳ650 bp) was purified,
The consequences of elevated homocysteine have been stud- digested with restriction enzymes BamHI and EcoRI, and
ied in a number of in vivo and cell culture models. In chick, high ligated into a BamHI/EcoRI-treated pGEX-2T expression vec-
homocysteine levels resulted in a reduced amount of fibrillin-2 tor (Amersham Biosciences). Primer synthesis and sequence
and microfibrils in the elastic lamina of the aorta (32). A defect confirmation were performed by The Centre for Applied
in fibrillin-1 deposition by smooth muscle cells was mechanis- Genomics at The Hospital for Sick Children. Expression and
tically linked to a deficiency in cysteine rather than to elevated purification of the polypeptide used techniques described pre-
homocysteine levels (33). Baumbach et al. (34) showed that the viously (27, 28). The identity of the polypeptide product was
structures of cerebral arterioles are altered in heterozygous confirmed by amino acid analysis and mass spectrometry, both
ϩ/Ϫ
Cbs
mice on a high methionine diet. These authors ob- performed by the Advanced Protein Technology Center at The
served a hypertrophy of cerebral arterioles resulting from an Hospital for Sick Children.
increase in the cross-sectional area of the arteriolar wall, attrib-
Preparation of Homocysteine and Homocysteinylation of
uted to an increase in the ratio of smooth muscle cells and Proteins—L-Homocysteine was prepared by reacting cyclic
elastin components versus the stiffer components like collagen L-homocysteine-thiolactone (Sigma) with 5 M NaOH using an
and basement membranes that remained constant. Recently, established procedure with minor modifications as described in
we and others showed that fragments of fibrillin-1 are a target detail elsewhere (35, 39). To prevent oxidation, aliquots were
for homocysteine (35, 36). The modified fragments became frozen immediately either on dry ice or in liquid nitrogen,
more susceptible to proteolysis, lost their ability to bind cal- stored at Ϫ80 °C, and re-thawed only once. The cysteine stock
cium, and demonstrated an altered secondary structure.
solution was prepared by dissolving L-cysteine hydrochloride
Here we demonstrate consequences of homocysteinylation on (Fluka) in the same buffer as the final buffer after homocysteine
functional properties of fibrillin-1 and tropoelastin. Homocystei- preparation (150 mM NaOH, 150 mM KH2PO4, pH ϳ 7). Ali-
nylation of fibrillin-1 led to alterations of self-interaction proper- quots were frozen and stored as described above for homocys-
ties. This was functionally attributed to a reduction of disulfide- teine. Homocysteine and cysteine used in cell culture assays
bonded C-terminal fibrillin-1 multimers. In a cell culture model, was sterile filtrated (0.22 ␮m) before aliquotation. To deter-
the deposition of a fibrillin-1 network was reduced after incuba- mine the concentration of free thiol groups in homocysteine
tion with homocysteine, but not with cysteine. Using elastin-like and cysteine solutions, Ellman reagent (5,5Ј-dithiobis 2-nitro-
polypeptides, we demonstrate a functional consequence of homo- benzoic acid, Fisher Scientific) was used according to the man-
cysteinylation on the coacervation properties. These data provide ufacturer’s protocol (40).
new important insights in potential pathogenetic mechanisms
To simulate pathological conditions, fibrillin-1 fragments
leading to the connective tissue phenotype observed in individuals rFBN1-N and rFBN1-C were incubated with 0–1000 ␮M homo-
with homocystinuria caused by CBS deficiency.
cysteine or with cysteine as a control at 37 °C for 24–72 h. After
12 h, a second aliquot of homocysteine or cysteine was added to
compensate for the relatively short half-life of homocysteine
EXPERIMENTAL PROCEDURES
Recombinant Fibrillin-1 Fragments and Elastin-like and cysteine due to oxidation. The proteins were dialyzed
Polypeptides—The N-terminal (rFBN1-N) and C-terminal against TBS including 2 mM CaCl2 and the concentration was
(rFBN1-C) halves of human fibrillin-1 were expressed with a determined using the BCA protein assay kit (Pierce). The integ-
hexahistidine tag at the C terminus in human embryonic kidney rity of the proteins was assessed using SDS-PAGE and Coomas-
cells (293, American Type Culture Collection) and purified sie Brilliant Blue staining (G-250, Fisher Scientific). To control
using Ni2ϩ-chelating chromatography as described previously for acidification in thiol concentrations above 500 ␮M, 1 M Tris
(35, 37, 38). Separation of rFBN1-C multimers from monomers (pH 7.4) was added to a final concentration of 33.3 mM.
was performed by gel filtration chromatography as described in
Solid Phase Binding Assays—The methodology for the solid
detail elsewhere (13). The purity of the proteins was assessed phase binding assays was previously described (41). Briefly, 1 ␮g
using SDS-PAGE under reducing conditions (20 mM dithio- of protein/well in TBS was coated on a 96-well Maxisorp flat
threitol (DTT)). Proteins were stored in 50 mM Tris, 150 mM bottom plate (Nalge Nunc International) overnight at 4 °C.
NaCl, pH 7.4 (TBS), including 2 mM CaCl2.
After blocking with 5% nonfat milk in TBS (blocking buffer), the
Expression, purification, and characterization of the elastin- soluble ligand was added in blocking buffer including 2 mM
like polypeptide EP20-24-24 containing the protein sequence CaCl2 either in 1:3 serial dilutions starting from 50 ␮g/ml, or in
encoded by exons 20-21-23-24-21-23-24 of human tropoelas- 1:2 serial dilutions starting from 100 ␮g/ml. Bound proteins
tin has been described previously (27). The construct for ex- were detected using a primary polyclonal rabbit antibody
pression of EP20-24-24/36, which includes additionally the against rFBN1-N or rFBN1-C (1:1000 diluted in blocking
protein sequence coded by exon 36 of human tropoelastin con- buffer) (42, 43), and a secondary horseradish peroxidase-con-
taining the two cysteine residues, was generated by a PCR strat- jugated antibody (Peroxidase AffiniPure goat anti-rabbit IgG,
egy using the EP20-24-24 plasmid as template and the following 1:800, Jackson ImmunoResearch Laboratories Inc.). The color
primers representing exon 36 and additional restriction sites reaction was performed using 1 mg/ml of 5-aminosalicylic acid
for cloning: BamHI-Ex20, 5Ј-CTGCTAGGGGGATCCAT- (Sigma) dissolved in 20 mM phosphate buffer (pH 6.8) including
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Homocysteinylation of Elastic Fiber Proteins
0.045% (v/v) hydrogen peroxide. The reaction was stopped with buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM
2 M NaOH and the absorbance at 492 nm was measured using a Na2HPO4, and 1.47 mM KH2PO4, pH 7.4, general washing
microplate reader (AD 340, Beckman Coulter). To compare buffer), the cells were fixed in ice-cold 70% methanol, 30% ace-
different sets of experiments, relative binding values were cal- tone. Cells were washed again, blocked for 60 min with PBS
culated by setting the absolute binding at the highest soluble including 10% normal goat serum (PBS-G) (Jackson Immu-
ligand concentration of the control to 100%. The curves were noResearch Laboratories, Inc.), and incubated with the primary
fitted using a hyperbolic model: A492 ϭ (P1 ϫ [L])/(P2 ϩ [L]) anti-fibrillin-1 ␣-rFBN1-C (1:1000) and anti-fibronectin FN-15
(Origin software version 7), where A492 represents the absorp- (Sigma, 1:1000) diluted in PBS-G for 2 h. After washing, the
tion at 492 nm, [L] the concentration of the soluble ligand in nM, cells were incubated for 2 h with fluorescein isothiocyanate-
P1 the overall binding, and P2 the dissociation constant (KD) in labeled goat anti-mouse (1:100) and Cy3-labeled goat anti-rab-
nanomolar. Statistical significance was calculated employing bit (1:200) secondary antibodies (Jackson ImmunoResearch
the two-sided Student’s t test. The solid phase binding assay Laboratories, Inc.) diluted in PBS-G. After washing with PBS
technique using antibodies as the detection method has restric- and one wash with distilled water, the nuclei of the cells were
tions in deducing absolute values for binding affinities. This is stained with 4Ј-6-diamidino-2-phenylindole (1 ␮g/ml in dis-
due to the non-linearity of the detection signals generated by tilled water; Invitrogen), washed again with water, cover-
primary and secondary antibodies (44). Therefore, the binding slipped, and examined using an Axioskop 2 microscope,
data after hyperbolic curve fitting provide apparent values, equipped with an Axiocam camera (Zeiss). Pictures were taken
which can be used to compare the effects of homocysteine and using AxioVision software version 3.1.2.1 (Zeiss).
cysteine on fibrillin-1 interaction properties. The term “overall
Dot-blot Analysis of Secreted Extracellular Matrix Proteins—
binding” refers to the value on the y axis obtained for the hori- The secretion of fibrillin-1 and fibronectin from HSFs into the
zontal asymptote after hyperbolic curve fitting, and the term culture medium was monitored by dot-blot analysis. 25 ␮l of
“apparent KD” refers to the concentration of soluble ligand in the medium of HSFs grown in the chamber slides for immuno-
nanomolar resulting in 50% overall binding. Note that the fluorescence experiments were diluted in 475 ␮l of TBS. The
apparent KD is reciprocal to the apparent affinity.
samples were blotted on a 0.45-␮m nitrocellulose membrane
To immobilize heparin derived from porcine intestinal (Bio-Rad) using a Bio-dot microfiltration apparatus (Bio-Rad).
mucosa (Sigma; product number H3393) on a 96-well plate, it The membrane was blocked with TBS including 5% nonfat milk
was covalently coupled to bovine serum albumin (BSA, Fisher for 1 h, followed by a 2-h incubation with the primary
Thermo Scientific, BP1605-100) using 1-ethyl-3-(3-dimethyl- ␣-rFBN1-C (1:1000) and FN-15 (1:1000) antibodies, diluted
aminopropyl)carbodiimide hydrochloride (Pierce) as a cross- in 5% BSA in TBS. After a 2-h incubation with either goat
linker. Aliquots of 0.2 ml of BSA (20 mg/ml in 0.1 M MES (pH anti-rabbit or goat anti-mouse horseradish peroxidase con-
4.7)) were mixed with 0.5 ml of heparin sodium salt (10 mg/ml jugate (both diluted 1:800 in TBS), the dot-blot was devel-
in 0.1 M MES, pH 4.7), added to 0.1 ml of 1-ethyl-3-(3-dimeth- oped in TBS, 17% methanol, including 0.02% (v/v) H2O2 and
ylaminopropyl)carbodiimide hydrochloride (10 mg/ml in 0.5 mg/ml of 4-chloro-1-naphthol.
water) and incubated for 2 h at 22 °C in an Eppendorf shaker
Quantitative Real Time-Polymerase Chain Reaction—30 ϫ
(900 rpm). To separate the cross-linked BSA-heparin from the 104 HSFs in DMEM were seeded on a 6-well plate (Costar,
non-reacted BSA and heparin, the reaction mixture was loaded Corning Inc.) in duplicates and after 24 h, the medium was
on a Superose 12 gel filtration column (GE Healthcare), equili- changed to DMEM including homocysteine or cysteine. The
brated in 0.1 M NaH2PO4, 900 mM NaCl (pH 7.2) at 0.5 ml/min. medium was changed every 12 h for 3 days. Total RNA was
Fractions of 1 ml were collected and tested for reactivity with extracted using the RNeasy Plus Mini Kit (Qiagen) as instructed
rFBN1-N and rFBN1-C by solid phase binding assay. Positive by the supplier. The same amounts of total RNA from each set
fractions were pooled, the protein concentration was deter- of samples was reverse-transcribed with random hexamer
mined using the BCA assay and stored at Ϫ20 °C.
primers and Superscript III (Invitrogen) using the manufactur-
Immunofluorescence—Primary human skin fibroblasts er’s protocol. Quantitative real time PCR analysis was per-
(HSFs) were isolated from the foreskins of healthy individuals formed in duplicates with TaqMan Gene Expression Assays
(2–5 years of age) after a standard circumcision procedure. The according to the manufacturer’s protocol (Applied Biosystems;
procedure was approved by the local ethics committee (PED- 7500 Fast Real-time PCR System). 2.5 ␮l of the cDNA was
06-054), and informed consent was obtained from the parents amplified using probes for human fibrillin-1 (Hs00171191_
of the tissue donors. HSFs were used for the experiments m1), fibronectin (Hs01549940_m1), and ribosomal 18S RNA
between passages 2 and 7 and grown at 37 °C in a 5% CO2 (HS99999901_s1) as internal control. Relative quantification
atmosphere with Dulbecco’s modified Eagle’s medium supple- was calculated by setting the signals from the non-treated sam-
mented with 2 mM glutamine, 100 units/ml of penicillin, 100 ple divided by the signal for the 18S RNA to 1 (Applied Biosys-
␮g/ml of streptomycin, and 10% fetal calf serum (DMEM; tems; 7500 System software version 1.3.0).
Wisent Inc.). For immunofluorescence experiments, cells were
Semiquantitative Measurement of Extracellular Matrix
seeded in 8-well chamber slides (7.5 ϫ 104 cells/well) and the Deposition—The amount of extracellular matrix secreted by
medium was changed after 24 h to DMEM containing homo- fibroblasts was determined in a microplate-based assay as pub-
cysteine or cysteine. For the next 3 days, the medium was lished with some modifications (45). HSFs were seeded in 96-
changed every 12 h to mimic a “long term” exposure to homo- well plates (Costar, Corning Inc.) at a density of 1.7 ϫ 104 cells/
cysteine or cysteine. Following a washing step with phosphate- well in 50 ␮l of DMEM in quadruplicates. After 24 h, the
1190 JOURNAL OF BIOLOGICAL CHEMISTRY
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Homocysteinylation of Elastic Fiber Proteins
medium was changed to DMEM including different concentra-
tions of homocysteine and cysteine and for the following 3 days,
the medium was changed every 12 h. For a background control,
four wells with 1.7 ϫ 104 cells/well in 50 ␮l of DMEM were
seeded 12 h before the end of the incubation period. This short
culture period is not sufficient to produce a fibrillin-1 network
and therefore, the cellular background can be assessed. The
cells with the extracellular matrix layer were washed with PBS
(general washing buffer) and fixed in 50 ␮l of ice-cold 70%
methanol, 30% acetone. Cells were washed again, blocked for 60
min with 75 ␮l of PBS-G, and incubated with 50 ␮l of the pri-
mary monoclonal fibrillin-1 F2 (1:1000) (10) or FN-15 (1:1000)
antibody diluted in PBS-G for 2 h. After washing, the cells were
incubated for 2 h with 50 ␮l of goat anti-rabbit (1:100) or goat
anti-mouse horseradish peroxidase conjugate (1:200) diluted in
PBS-G. After washing with PBS the color reaction was per-
formed as described under “Solid Phase Binding Assays” and
the absorption at 492 nm was measured.
FIGURE 1. Fibrillin-1 fragments used in this study. A, schematic overview of
the two recombinant halves of fibrillin-1, rFBN1-N (N terminus) and rFBN1-C
(C terminus). TB/8Cys, transforming growth factor-␤ binding protein-like
Analysis of Coacervation—The coacervation experiments
were carried out as described in detail elsewhere (46). For our module/8 cysteine motif. B, the fragments were either not treated (Ϫ) or
treated with 300 ␮M homocysteine (Hcy) or cysteine (Cys), and aliquots of 10
␮g were then separated by 7.5% SDS-PAGE under reducing conditions (20
mM DTT). There are no major differences detectable between the modified
and non-modified fragments, notably no degradation. Positions of molecular
studies, we used polypeptides EP20-24-24/36 including the two
cysteines in the sequence encoded by exon 36 and EP20-24-24
without the exon 36-encoded protein sequence as a control
marker proteins are indicated in kilodaltons.
(27). 25 ␮M polypeptide in 50 mM Tris (pH 7.2), 1.5 M NaCl was
incubated without (control), with 300 ␮M homocysteine, or
with 300 ␮M cysteine for 1 h on ice prior to the coacervation exposure of extracellular proteins to lower concentrations of
experiment. Coacervation was measured at 440 nm in a Shi- homocysteine, found in patients with various forms of homo-
madzu UV-2401PC UV-visible recording spectrophotometer cysteinemia (3). Fig. 1B shows that there is no degradation
with a temperature and stir rate controller (Mandel Scientific). observed for rFBN1-N and rFBN1-C after modification with
The initial temperature was set 5 °C below the coacervation cysteine or homocysteine as judged by SDS-PAGE analysis
temperature (Tc) and raised by 1 °C/min to a final target tem- under reducing conditions. Occasionally, rFBN1-N but not
perature 3–4 °C above Tc. The final temperature was kept con- rFBN1-C tended to precipitate after homocysteinylation.
stant for 40 min to record the maturation phase. The tempera-
Fibrillin-1 N- to C-terminal Self-interaction—We used a well
ture ramps were 24–35 °C for EP20-24-24 and 25–37 °C for established solid phase binding assay to address the question, if
EP20-24-24/36. Tc was noted as the onset of the increase in the N- to C-terminal self-interaction of fibrillin-1 is affected by
absorption and the results of the experiments are expressed as a homocysteinylation. This self-interaction is considered one of
shift of the coacervation temperature as compared with the the early steps in the biogenesis of microfibrils. In this assay, the
control (⌬Tc). The curves were fitted using the MathWork soft- N-terminal half (rFBN1-N) was coated and the C-terminal half
ware (Natick, MA) with the equation: A ϭ Ϫa ϫ e(Ϫk ϫt) ϩ b ϫ (rFBN1-C) was added as soluble ligand in serial dilutions. Fig. 2
c
e(Ϫk ϫt) ϩ c, where A is the absorption at 440 nm, a, b, and c shows that homocysteinylation of rFBN1-N (Fig. 2A) and
m
are proportionality factors, kc is the rate constant for the coac- rFBN1-C (Fig. 2B) lowers the apparent affinity and the overall
ervation, and km is the rate constant for the maturation. The binding capacity to the respective counterpart. It clearly dem-
velocities for the coacervation and maturation were calculated onstrates that homocysteinylation is interfering with the capa-
as Vc ϭ a ϫ kc and Vm ϭ b ϫ km. Details of this method are bility of the N and C terminus to interact with each other. This
explained in Ref. 46.
effect is specific for homocysteine, because modification with
cysteine does not result in a significant change in the N- to
C-terminal binding of rFBN1-C to rFBN1-N. The homocystei-
RESULTS
Homocysteinylation of Fibrillin-1 Fragments—The protein nylation of the N terminus tends to more strongly affect the
fragments used in this study are well characterized N- and overall binding (reduction to 72.9% of the control) than the
C-terminal halves (rFBN1-N and rFBN1-C, respectively), span- apparent affinity to the C terminus (ϳ1.2-fold higher KD) (Fig.
ning the entire fibrillin-1 molecule (Fig. 1A). Because the inter- 2C). However, the difference did not reach statistical signifi-
action repertoire of these molecules was studied extensively, cance. In contrast, the modification of rFBN1-C with homocys-
they serve as valuable tools to elucidate mechanistic aspects of teine led to a dramatically increased apparent KD indicating a
fibrillin-1/microfibril assembly. The fragments were incubated lower affinity for the N-terminal half of fibrillin-1 (ϳ12-fold
with 300 ␮M homocysteine or cysteine for 24 h. This is in the lower) and reduces the overall binding to 81.9% of the control
pathophysiological range of concentrations detected in the (Fig. 2D). The strong increase in the apparent KD of the binding
plasma of patients with severe forms of homocystinuria caused of the homocysteinylated C terminus to the N terminus (p ϭ
by CBS deficiency. In addition, this mimics the continuous 0.046, Fig. 2D) and the reduction in binding of the homocys-
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Homocysteinylation of Elastic Fiber Proteins
FIGURE 3. Homocysteinylation of N- and C-terminal fibrillin-1 fragments
results in a complete loss of self-interaction. N and C termini of fibrillin-1
were homocysteinylated (300 ␮M) and tested in a solid phase binding assay
using immobilized rFBN1-N and soluble rFBN1-C. The control showed the
expected hyperbolic binding curve (rFBN1-N/rFBN1-C, f). Modification of
both binding ligands with homocysteine (*) resulted in an almost complete
loss of the ability to self-interact (rFBN1-N*/rFBN1-C*, ꢀ). Representative data
are shown as mean Ϯ S.D. values of duplicates.
teinylated C terminus to the N terminus (p ϭ 0.028, Fig. 2D) are
statistically significant. To simulate the effect of homocysteine
on full-length fibrillin-1, we performed a solid phase binding
assay using simultaneously both, homocysteinylated N and C
termini (Fig. 3). Modification of both fibrillin-1 termini resulted
in an almost complete reduction of the self-interaction as com-
pared with the homocysteinylation of only one ligand (compare
Figs. 2, A and B, with 3). These data indicate that both self-
interaction sites, on the N and C terminus, are functionally
affected by homocysteinylation.
Multimerization of the C Terminus Is Reduced by Homo-
cysteinylation—Recently, we showed that disulfide bond-medi-
ated multimerization of the C terminus of fibrillin-1 is a neces-
sary prerequisite for the N- to C-terminal self-interaction (13).
To test if multimerization of the C terminus is affected by homo-
cysteine, we separated multimers from monomers using gel
filtration. After incubation with homocysteine or cysteine, the
multimers were subjected to SDS-PAGE under non-reducing
conditions and silver-stained (Fig. 4, A–C). The amount of mul-
timers was reduced at homocysteine concentrations of 300 ␮M
and higher. Full reduction is achieved using 20 mM DTT, which
served as a control (Fig. 4A, DTT). The monomers resulting
from homocysteinylation appear to migrate at a lower apparent
molecular weight correlating with the homocysteine concen-
tration in the sample (Fig. 4A, arrowheads). The consequence of
homocysteine on the multimerization of the fibrillin-1 C termi-
nus is significantly more specific for homocysteine because cys-
teine in the same concentration range only had a little effect
(Fig. 4B). Only a very faint protein band of the monomeric size
appears at a cysteine concentration of 1 mM (Fig. 4B, arrow-
FIGURE 2. Fibrillin-1 N- to C-terminal self-interaction is impaired by
homocysteinylation. Solid phase binding assays with immobilized rFBN1-N
and soluble rFBN1-C were used to assess the effect of homocysteinylation
on the N- to C-terminal self-interaction process. A, consequences on the inter-
action between modified rFBN1-N with non-modified rFBN1-C using homo-
cysteinylated (300 ␮M Hcy, F), cysteinylated (300 ␮M Cys, Œ), or non-treated
(control,f)rFBN1-N(nϭ2).B,consequencesonthebindingofmodifiedrFBN1-C
to non-modified rFBN1-N, using homocysteinylated (300 ␮M Hcy, E), cysteiny-
lated (300 ␮M Cys, ‚), or non-treated (control, Ⅺ) rFBN1-C (n ϭ 4). Notice that
there remains always one binding partner unmodified in both panels. In Aand B,
data are shown as percentage of the highest soluble ligand concentration of the
non-treated control and represent mean Ϯ S.D. The signal at 492 nm for the
controls was in all experiments Ͼ1.0 absorption units. Cand D, statistical analysis
of data shown in A and B for the overall binding and the apparent KD of N- to
C-terminal self-interactions with modified rFBN1-N (C) or rFBN1-C (D). The bars
represent mean Ϯ S.D. of the overall binding (%) derived from the horizontal heads). This band migrated slightly faster then the fragment
asymptote of the hyperbolic fit of individual experiments and the apparent KD
reduced with DTT, but not as fast as the homocysteine-modi-
(nM) representing the ligand concentration at 50% overall binding. The statistical
significance was calculated using the two-sided Student’s t test. p values Ͻ0.05
are considered statistically significant.
fied fragment (Fig. 4C). Analyzing the same set of samples after
modification with 1 mM homocysteine, cysteine, or DTT sub-
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Homocysteinylation of Elastic Fiber Proteins
FIGURE4. Homocysteinereducestheamountoffibrillin-1C-terminalmultimersanddoseandtimedependentlyaffectsthefibrillin-1self-interaction.
Multimers of rFBN1-C were purified using gel filtration and incubated with different concentrations of homocysteine (A, Hcy) and cysteine (B, Cys), as indicated
in micromolar. Shown are silver-stained gels after non-reducing SDS-PAGE (4–20%). Incubation with 1 mM DTT served as controls. Monomers of rFBN1-C are
indicated by arrowheads. Arrows indicate the border between stacking and separating gels. C, multimers of rFBN1-C were first incubated with 1 mM homocys-
teine(Hcy), cysteine(Cys), orDTTcomparedwithanon-treatedcontrolandthenseparatedby4–20%gradientSDS-PAGEundereithernon-reducedorreduced
(additional 20 mM DTT) conditions as indicated. Note the qualitative difference between the homocysteinylated proteins analyzed under reduced versus
non-reduced conditions. Positions of molecular marker proteins (M) are indicated in kilodaltons. D, solid phase binding assay of soluble rFBN1-C (734 nM)
homocysteinylated with 50–1000 ␮M as indicated and coated non-modified rFBN1-N. The bars represent the relative binding compared with the control with
non-modified rFBN1-C (100% binding). Statistically significant differences compared with the control are indicated (p Յ 0.05 level). E, rFBN-1C was homocys-
teinylated with 300 and 1000 ␮M homocysteine for 24, 48, and 72 h as indicated, and the N- to C-terminal fibrillin-1 self-interaction was analyzed as in D with
734 nM soluble rFBN1-C. The relative absorption for each time point is blotted compared with non-treated controls (100% binding). Data are shown as mean Ϯ
S.D. values of duplicates.
sequently under reducing conditions (20 mM DTT) resulted in cysteine concentrations (up to 1000 ␮M) to simulate the long
bands migrating at identical molecular weight positions (Fig. term exposure to lower homocysteine concentrations, as it
4C). This result indicates that the apparent lower molecular occurs in patients.
weight is caused by reversible modifications of the proteins. In
Binding of Homocysteinylated Fibrillin Fragments to Heparin—
the self-interaction assay, the reduction in binding of the homo- Fibrillin-1 interacts with heparin and heparan sulfate through
cysteinylated C terminus of fibrillin-1 to the N terminus is dose- several domains distributed throughout the molecule, includ-
dependent with respect to the homocysteine concentration ing the N and C terminus (43, 47, 48). Therefore, we tested if
used for modification (Fig. 4D). This correlates with the dose homocysteinylation interferes with binding of fibrillin-1 frag-
dependence observed for the homocysteine-induced reduction ments to heparin (Fig. 5). Homocysteinylation of rFBN1-N and
of the C-terminal multimers. To analyze the time course of rFBN1-C resulted in a significant decrease in binding to hepa-
fibrillin-1 homocysteinylation, we incubated the rFBN1-C frag- rin. The modification with cysteine also reduced the binding
ment with different concentrations of homocysteine for various capability to a similar extent and the differences between the
time points and tested self-interaction with non-modified two types of modifications were not statistically significant.
rFBN1-N (Fig. 4E). The 72-h incubation with 300 ␮M homocys- The observation of nonspecific consequences of homocystei-
teine resulted in a similar reduction of the self-interaction than nylation compared with cysteinylation strengthens the rele-
the 24-h incubation with 1000 ␮M. Therefore, it is possible to vance of the homocysteine-specific effects observed for the
use short term exposure of fibrillin-1 fragments with high homo- fibrillin-1 N- to C-terminal self-interaction described above.
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Homocysteinylation of Elastic Fiber Proteins
cysteine in reducing fibrillin-1 deposition by cells. Analysis of
secreted fibrillin-1 and fibronectin in the cell culture medium
by dot-blotting showed no differences between controls and
homocysteine or cysteine-treated samples (Fig. 6D). We con-
clude that reduced formation of the fibrillin-1 network after
incubation with homocysteine represents a deposition defect
rather than deficient transcription or secretion.
Coacervation Properties of Elastin-like Polypeptides Are
Altered upon Homocysteinylation—To assess the effect of homo-
cysteine on elastin-like polypeptides, we used EP20-24-24 and
EP20-24-24/36 containing the protein sequence encoded by
exon 36 with the sole two cysteines found in human tropoelas-
tin (Fig. 7A). The cysteines are engaged in an intramolecular
disulfide bond (24). After homocysteinylation only, we
observed a statistically significant increase in the coacervation
temperature of 1.4 °C as compared with the control (p ϭ 0.003,
Fig. 7B). This was not observed after incubation with cysteine,
ruling out an effect of the sole presence of a thiol group con-
taining reagent and demonstrating the specificity for homocys-
teine. The coacervation temperature of the control polypeptide
EP20-24-24 did not change significantly after incubation with
either homocysteine or cysteine. Interestingly, the velocity for
the coacervation and the maturation stage does not change in
the presence of homocysteine (Fig. 7C). This is different as
compared with other factors affecting the coacervation temper-
ature, such as salt and other proteins of the elastic fiber system,
which also affect either the velocity of coacervation or matura-
tion, or both (27, 46).
FIGURE 5. Homocysteinylation and cysteinylation affect binding of the
fibrillin-1 N and C terminus to heparin. Solid phase binding assays were
used to assess the effect of homocysteinylation of rFBN1-N and rFBN1-C on
theinteractionwithBSA-heparin. SolublerFBN1-NandrFBN1-C(both734nM)
were used as soluble ligands and BSA-heparin as immobilized ligand. Homo-
cysteinylation (300 ␮M) and cysteinylation (300 ␮M) reduced the binding to
BSA-heparin compared with non-modified rFBN1-N and non-modified
rFBN1-C (controls, 100%). Representative data are shown as mean Ϯ S.D.
valuesofthreeexperiments. Theoriginalsignalat492nmforthecontrolswas
in all experiments between 0.4 and 1.0 absorption units. The significance was
calculated using the two-sided Student’s t test.
Deposition of Fibrillin-1 by HSFs Is Reduced in the Presence of
Homocysteine—To assess the effect of homocysteine on the
deposition of fibrillin-1 into the extracellular matrix, HSFs were
DISCUSSION
The genetic disorder homocystinuria caused by CBS defi-
grown in the presence of 1000 ␮M homocysteine or cysteine for ciency and Marfan syndrome overlap in some clinical symp-
4 days and analyzed by immunofluorescence. The relatively toms, primarily in the skeletal system and the eye. This led
high homocysteine concentration was required because the originally to the hypothesis that fibrillin-1 is a target for homo-
concentration of free (active) homocysteine/cysteine is reduced cysteine, which has been confirmed by us and others (35, 36).
in the presence of serum components such as BSA (49). The For the work presented here, we asked the question, does
matrix deposition of fibrillin-1 was always significantly reduced homocysteinylation affect the function of components of the
after treatment with homocysteine, but not with cysteine, microfibril/elastic fiber system, including fibrillin-1 and tro-
whereas the deposition of fibronectin was not affected (Fig. 6A). poelastin. To assess effects of homocysteinylation on fibrillin-1
A semi-quantitative analysis of the fibrillin-1 and fibronectin self-interaction, correctly folded and well characterized N- and
network formation after treatment with homocysteine showed C-terminal halves of fibrillin-1 were utilized (38, 52). Studies
a significant and dose-dependent reduction for the fibrillin-1 with full-length fibrillin are currently hampered by its propen-
network, but not for the fibronectin network (Fig. 6B). No sig- sity to aggregate (15). A second aspect of this project focused on
nificant reduction could be detected with this assay for the the consequences of homocysteinylation on the disulfide bond
fibrillin-1 matrix deposited by HSFs in the presence of cysteine. in tropoelastin as assessed by an elastin-like polypeptide, which
To additionally test whether other metabolites altered in includes the two disulfide bond-forming cysteines located in
homocystinuria patients influence the fibrillin-1 network for- domain 36 of human tropoelastin (24).
mation, HSFs were treated with reduced cysteine (50 versus 300
We demonstrate here that homocysteinylation reduced
␮M for the control) and high methionine (1 mM versus 30–40 significantly N- to C-terminal fibrillin-1 self-interaction
␮M for the control) (50). No changes in the fibrillin-1 network properties, which represent a critical step in the biogenesis
formation were detected under these conditions (data not of microfibrils. Furthermore, homocysteinylation affected the
shown).
multimerization of the fibrillin-1 C terminus. Both effects were
Examination of mRNA levels by quantitative real time PCR specific for homocysteine as compared with cysteine. Recently,
showed a slight decrease of fibrillin-1 (16.8%) and fibronectin we demonstrated that the affinity of the fibrillin-1 N- to C-ter-
(8.3%) expression after homocysteine treatment, and a stronger minal self-interaction is strictly dependent on the multimeriza-
reduction in fibrillin-1 (35.4%) and fibronectin (31.1%) expres- tion state of the C terminus, ranging from very low affinity for
sion after incubation with cysteine (Fig. 6C). These slightly monomers to intermediate affinities for oligomers with 3–7
reduced mRNA levels do not explain the specificity of homo- subunits and to very high affinity for multimers with 8–12 sub-
1194 JOURNAL OF BIOLOGICAL CHEMISTRY
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Homocysteinylation of Elastic Fiber Proteins
FIGURE 6. Functional consequence of homocysteine and cysteine on the fibrillin-1 network formation in cell culture. HSFs were used to study the effect
of homocysteine and cysteine on the formation of the extracellular fibrillin-1 network. A, immunofluorescence of fibrillin-1 (red) and fibronectin (green) after
incubation with 1000 ␮M homocysteine (Hcy) or cysteine (Cys) for 4 days. Note that the fibrillin-1 network is greatly reduced only in the presence of homocys-
teine. The experiments were repeated with cells from two different donors with identical results. The scale bar represents 100 ␮m. B, quantification of fibrillin-1
(black bars) and fibronectin (white bars) deposition by a microplate-based assay (see “Experimental Procedures”). Bars represent mean Ϯ S.D. values of 4 data
points. The statistical significance was calculated with a two-sided Student’s t test and the p values are indicated. C, to control for gene expression, quantitative
real time PCR with TaqMan probes for human FBN1 and FN was performed after growing the fibroblasts for 4 days in the presence of 1000 ␮M homocysteine
or cysteine. Ribosomal 18S RNA was used as an internal standard. The asterisk indicates a statistically significant difference compared with the respective
control. D, the cell culture medium (25 ␮l) from the final 12-h incubation period of the experiment shown in A was analyzed by dot-blotting using specific
antibodies against fibrillin-1 and fibronectin as indicated. Notice that the amount of secreted soluble fibrillin-1 or fibronectin is similar in all samples.
units (13). In that study, multimerization was shown to be higher nucleophilic reactivity of homocysteine compared with
mediated by solvent-exposed intermolecular disulfide bonds. cysteine, because cysteine in identical concentrations was non-
In light of this, we conclude from our data presented here that reactive. It is likely that homocysteine reduces accessible cys-
homocysteine interferes with intermolecular disulfide bonds teine-cysteine bonds and forms mixed cysteine-homocysteine
stabilizing the high molecular weight assemblies mediated disulfides. This notion is further supported by the fact that
through the C terminus, and leading to the loss of apparent homocysteine-modified rFBN1-C monomers, which migrate
affinity to the N terminus. This modification requires the slightly faster in polyacrylamide gel electrophoresis than the
JANUARY 8, 2010•VOLUME 285•NUMBER 2
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Homocysteinylation of Elastic Fiber Proteins
non-homocysteinylated monomers, adopt a normal migration
pattern after additional reduction with dithiothreitol. In addi-
tion to the above discussed mechanism, the following factors
might also mechanistically contribute to the loss of N- to C-ter-
minal self-interaction properties. Homocysteinylation of fibril-
lin-1 led to subtle structural changes in cbEGF domains (35). In
this regard, structural modifications in cbEGF41-43, which has
been shown to contain the binding domain for the N terminus,
may be a critical factor (13). We and others have previously
shown that the N- to C-terminal fibrillin-1 self-interaction is
calcium dependent (14, 15), and that homocysteinylation abol-
ishes calcium binding to cbEGF domains in fibrillin-1 (35).
With this background, homocysteinylation of the N- or C-ter-
minal half might affect their calcium-binding properties
required for self-assembly. In addition, it is possible that homo-
cysteine modifies the non-paired cysteine residue in the first
hybrid domain of the fibrillin-1 N terminus necessary for the
self-assembly process (53). Given the additive effect when both,
the fibrillin-1 N and C terminus, are homocysteinylated, we
suggest that a combination of the above described mechanisms
is likely to lead to the observed functional consequences.
In cell culture the deposition of fibrillin-1 is reduced in the
presence of homocysteine, but not in the presence of cysteine.
This clearly demonstrates that the observed reduction in fibril-
lin-1 N- to C-terminal self-interaction upon homocysteinyla-
tion translates into a reduced network formation in cell culture.
It was previously shown using smooth muscle cells that 300 ␮M
homocysteine did not reduce the deposition of fibrillin-1 (33).
We observed with fibroblasts a marked reduction of the fibril-
lin-1 network formation with 1000 ␮M homocysteine, but not
with 300 ␮M. This suggests a threshold effect that could be
attributed to the serum present in the culture medium required
for a proper fibrillin-1 deposition. Albumin is a target for homo-
cysteine and its abundance in serum could reduce the effective
homocysteine concentration (49, 57). In addition, a threshold
effect for homocysteine has been recently described in a Cbs-
deficient mouse model (59).
Giusti et al. (54) recently demonstrated a positive correlation
of the severity of cardiovascular manifestations, including aor-
tic involvement, with mildly elevated homocysteine levels in
individuals with Marfan syndrome. Aortic aneurysms are a
common symptom in Marfan syndrome and can lead to death.
In light of this, it is possible that mutations in fibrillin-1 further
contribute to its susceptibility for homocysteinylation poten-
tially enhancing the above discussed molecular consequences
on fibrillin-1 self-assembly and thus further contributing to the
pathogenetic sequence.
Fibrillin-1 has been shown to interact with heparin/heparan
sulfate in various regions of fibrillin-1 (43, 47, 48). Here, we
show that homocysteinylation of fibrillin-1 reduces heparin
binding to fibrillin-1, similar to cysteinylation. In this case, the
reactive nucleophilic potential of the cysteine sulfhydryl group
FIGURE 7. Effect of homocysteine on the coacervation and maturation of
elastin-like polypeptides. Coacervation was used to study the effect of
homocysteine on elastin-like polypeptides. A, schematic overview of the
domain structure of human tropoelastin and the two elastin-like polypep-
tides EP20-24-24 and EP20-24-24/36. Relevant domain numbers are indi-
cated. B, analysis of the coacervation temperature after incubation of the
polypeptides with 300 ␮M homocysteine (Hcy) or 300 ␮M cysteine (Cys). The
coacervation temperatures are indicated as ⌬Tc compared with the coacer-
vation temperature of non-modified elastin-like peptides. Tc for EP20-24- t test. C, analysis of the velocities of coacervation (Vcoac, black bars) and mat-
24 ϭ 28.7 °C (Ϯ0.3 °C) and EP20-24-24/36 ϭ 28.6 °C (Ϯ0.8 °C). Note that only uration (Vmat, white bars) after modification with homocysteine (Hcy) and cys-
modification with homocysteine significantly increased the coacervation teine(Cys)areindicatedin⌬Aat440nm/min. Theexperimentswererepeated
temperature (ϩ1.4 °C) of EP20-24-24/36, but not modification with cysteine 3–5 times with different batches of polypeptides. The mean Ϯ S.D. are
(ϩ0.1 °C). Indicated p values were calculated using a two-sided Student’s indicated.
1196 JOURNAL OF BIOLOGICAL CHEMISTRY
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Homocysteinylation of Elastic Fiber Proteins
is sufficient to modify intra- or inter-molecular disulfide bonds nective tissue phenotypes in CBS-deficient homocystinuria.
in heparin/heparan sulfate-binding regions of fibrillin-1. These Moreover, for the first time we provided in vitro evidence that
results exemplify that the different disulfide bonds in fibrillin-1 tropoelastin is a target for homocysteinylation.
vary in their susceptibility for reduction by thiol-containing
compounds. A molecular targeting hypothesis was recently
developed stating that homocysteine attacks not all but only
specific disulfide bonds or free cysteines in proteins (55, 56). To
render free cysteines as targets for homocysteinylation, they
have to be solvent exposed and must have a relatively low pKa
value (Ͻ8.15), as defined by the sequence context of a given
protein. Disulfide bonds have to be solvent accessible and con-
tain high energy as estimated from the relative angles of the two
individual cysteines forming the disulfide bond, expressed as
the dihedral strain energy (Ͼ17.8 kJ/mol). In that study, cbEGF
modules 32–34 of fibrillin-1 were a predicted target for homo-
cysteinylation, which correlates with experimental data (36).
Given the large number of disulfide bonds in fibrillin-1, this
prediction algorithm may prove useful in combination with our
experimental data to identify potential cysteine target residues
for homocysteinylation and design new and testable hypotheses
for the pathogenetic mechanisms involved in Marfan syndrome
and homocystinuria.
Acknowledgment—We thank Dr. Lynn Sakai (Shriners Hospital for
Children, Portland, OR) for generously providing the F2 anti-fibril-
lin-1 monoclonal antibody. We are grateful to Dr. Jean-Martin
Laberge (Montreal Children’s Hospital) for providing foreskin
samples.
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1198 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 2•JANUARY 8, 2010

Glycobiology and Extracellular Matrices:
Functional Consequences of
Homocysteinylation of the Elastic Fiber
Proteins Fibrillin-1 and Tropoelastin
Dirk Hubmacher, Judith T. Cirulis, Ming
Miao, Fred W. Keeley and Dieter P. Reinhardt
J. Biol. Chem. 2010, 285:1188-1198.
doi: 10.1074/jbc.M109.021246 originally published online November 4, 2009
Access the most updated version of this article at doi: 10.1074/jbc.M109.021246
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