(Received for publication, July 17, 1995; and in revised form, October 9, 1995)
From the
Together with the 31-kDa microfibril-associated glycoprotein
(MAGP), four polypeptides designated MP340 (340 kDa), MP78 (78 kDa),
MP70 (70 kDa), and MP25 (25 kDa) have previously been identified in
tissue extracts designed specifically to solubilize the microfibrillar
component of elastic fibers. In the present study, both MP78 and MP70
were shown to be forms of a protein which is closely related to the
human protein ig-h3, and MP340 was confirmed to be the bovine form
of fibrillin-1. Peptide sequences from MP25 proved to be unique, and
affinity-purified anti-MP25 antibodies were shown, by
immunofluorescence and immunoelectron microscopy, to localize
specifically to the elastin-associated microfibrils. This confirmed
that MP25 was a distinct component of these structures. Expression
screening of nuchal ligament cDNA libraries yielded a cDNA, cM10A (770
base pairs) which encodes amino acid sequences matching those of the
MP25 peptides. Further library screening with cM10A identified cDNAs
which encode the complete primary structures of bovine and human MP25.
Bovine and human MP25 were found to be around 80% homologous and
contain 170 and 173 amino acids, respectively. Data base searches
revealed that MP25 had significant similarity of structure only with
MAGP, indicating that the two proteins form a new family of
microfibrillar proteins. In acknowledgment, MP25 has been formally
renamed MAGP-2, and MAGP is referred to as MAGP-1. The close similarity
between the two proteins (57%) is confined to a central region of 60
amino acids where there is precise alignment of 7 cysteine residues.
Elsewhere the MAGP-2 molecule is rich in serine and threonine residues
and contains an RGD motif. MAGP-2 lacks the proline-, glutamine-, and
tyrosine-rich sequences and a hydrophobic carboxyl terminus,
characteristic of MAGP-1. These structural differences suggest that
MAGP-2 has some functions which are distinct from those of MAGP-1. The
locus of the human MAGP-2 gene was identified on chromosome 12 in the
region of 12p12.3-12p13.1.
Considerable interest has centered recently on microfibrils,
10-12 nm in diameter, which occur in the extracellular matrix of
a diverse range of tissues. Parallel arrays of 12-nm microfibrils are
found as components of elastic fibers in tissues such as arteries,
lung, and some ligaments. These bundles of microfibrils appear to act
as templates for the deposition of tropoelastin during elastin
formation. Morphologically indistinguishable microfibrils also occur as
elastin-free bundles in tissues, such as the ciliary zonule of the eye,
periodontal ligament, skeletal muscle, and kidney, where they serve an
anchoring function. In other tissues, such as skin, some microfibrillar
bundles become associated with elastin, whereas others remain
elastin-free. The reasons for these tissue differences are unclear, but
they may be due, at least in part, to variations in the molecular
composition of the microfibrils and associated
proteins(1, 2, 3, 4, 5) .
Several proteins have been identified which appear to be structural
components of both elastin-associated and elastin-free microfibrils.
These include the 350-kDa glycoproteins fibrillin-1 and fibrillin-2 and
the 31-kDa, elastin-binding protein, MAGP ()(6, 7, 8, 9, 10, 11, 12) .
Fibrillin-1 and fibrillin-2 have been linked to the congenital
connective tissue disorders Marfan syndrome and congenital contractural
arachnodactyly, respectively(13, 14, 15) .
Both disorders are characterized by major skeletal and ocular defects,
but cardiovascular problems associated with Marfan syndrome are usually
lacking in patients with congenital contractural
arachnodactyly(16) . This pointed to developmental and tissue
differences in the expression of the two fibrillins, and this has been
confirmed by recent studies(11, 12, 17) . In
some tissues, such as elastic ear cartilage, fibrillin-1 and
fibrillin-2 were found by immunohistochemistry and in situ hybridization to have very different distribution patterns (12) . The findings led these authors to propose that
fibrillin-1-containing microfibrils provide long-term force-bearing
structural support whereas fibrillin-2-containing microfibrils regulate
the early process of elastic fiber assembly. However, there appears to
be extensive overlap of expression of the two proteins during the
development of a number of
tissues(11, 12, 17) . This suggests that
variations in other microfibril-associated proteins may also be
important for tissue-specific functions of the different groups of
12-nm microfibrils.
We have previously reported the preliminary
characterization of the major proteins extracted from the developing
elastic tissue, fetal bovine nuchal ligament, using a reductive saline
treatment. This reductive saline treatment, when preceded by exhaustive
extraction with the chaotrope guanidinium chloride, is relatively
specific for the solubilization of microfibrillar proteins which
otherwise would remain insoluble due to extensive intermolecular
disulfide bonding(18) . In addition to MAGP (31 kDa), which we
had previously characterized(7, 19) , this treatment
extracted four polypeptides with apparent molecular masses of 340, 78,
70, and 25 kDa which were named MP340, MP78, MP70, and MP25,
respectively. In the present report we describe the further
characterization of these species. MP340 was identified as bovine
fibrillin-1 by peptide sequencing. MP78 and MP70 were shown by peptide
mapping to be isoforms of a single protein. Sequencing of the peptides
indicated that the protein is closely related to the recently cloned
but poorly characterized human protein, ig-h3(20) .
Peptide sequencing of the 25-kDa species (MP25) indicated that it is a
distinct protein which we have now cloned. Herein we report that MP25
immunolocalizes specifically to elastin-associated microfibrils in
developing elastic tissue and that the protein has structural
similarities to MAGP. Thus we have renamed MP25 as MAGP-2 and now refer
to MAGP as MAGP-1.
For immunoelectron microscopy,
blocks (1 mm) of nuchal ligament tissue from a 240-day-old
fetal calf were fixed and embedded in LR White resin. Thin sections
were then cut and collected on Celloidon-coated nickel grids as
described previously(18) . The sections were treated with
phosphate-buffered saline containing 20 mM glycine (5 times
for 10 min each), and immunolabeling was performed as described
previously(23) . Anti-MAGP-2 antibodies, anti-tropoelastin
antibodies, and control serum were applied at the concentrations
described above, and binding was detected by protein A coupled to 10-nm
gold particles.
Figure 1: Comparison of CNBr peptide digests of MP78 and MP70. MP78 and MP70 were individually purified from a mixture of the two proteins by electrophoresis on 4% agarose gels and treated with CNBr to yield peptides for mapping as described in the text. Panel A, SDS-PAGE analysis on 10% gels of the purified proteins stained with Coomassie Blue. Lane 1, MP78 and MP70 before separation; lane 2, purified MP78; lane 3, purified MP70. Panel B, the CNBr digests analyzed by SDS-PAGE on 20% gels and silver stained. Lane 4, digest of MP78; lane 5, digest of MP70.
Figure 2:
Comparison of amino acid sequences. A, amino acid sequences encoded in human ig-h3 cDNA (20) (above) compared with sequences from MP78 and
MP70 peptides (below). B, a unique amino acid
sequence encoded in human fibrillin-1 cDNA (10) (above) compared with a sequence from a MP340
peptide (below). The location of each sequence within the
molecule is indicated by the residue numbers shown above. Vertical
lines indicate identity within the sequences. X denotes
an amino acid assignment which could not be clearly
designated.
Figure 3:
Immunoblotting of microfibrillar proteins
with affinity-purified anti-MAGP-2 antibodies. Panel A, a
typical reductive saline extract of fetal nuchal ligament analyzed by
SDS-PAGE on a 10% gel and stained with Coomassie Blue. Panel
B, a similarly stained 12% gel of the major proteins purified from
such extracts. Panel C, a matching immunoblot stained with
anti-MAGP-2 antibodies. Lane 1, MP340 (fibrillin-1); lane
2, MP78/70 (ig-h3); lane 3, MAGP-1; and lane
4, MAGP-2 (MP25).
Figure 4:
Immunofluorescence localization of
anti-MAGP-2 antibodies to elastic fibers. Unfixed sections of nuchal
ligament from a 210-day-old fetal calf were stained with anti-MAGP-2
antibodies (Panel A) and anti-tropoelastin antibodies (Panel B) as described in the text. Magnification,
170
Figure 5:
Immunoelectron microscopic localization of
anti-MAGP-2 antibodies to elastin-associated microfibrils. Sections of
nuchal ligament from a 240-day-old fetal calf were fixed, embedded, and
labeled using the immunogold technique as described in the text. Panel A shows parts of two elastic fibers stained with
anti-MAGP-2 antibodies. Each fiber is identified by microfibrils
(indicated by arrows) surrounding an amorphous core of elastin (e). Also indicated are electron-dense inclusions considered
to contain microfibrils embedded within the elastin core (arrowheads). A cell process (ce) separates the two
fibers. Panel B, control staining of an elastic fiber using
IgG from preimmune rabbit serum. Panel C, an elastic fiber
stained with anti-tropoelastin antibodies. Magnification,
45,000; bar = 0.1 µm.
Figure 6:
Identification of MAGP-2 mRNA.
Poly(A) RNA from developing nuchal ligament was
electrophoresed on a 1% agarose gel (1 µg/lane), Northern blotted,
and hybridized with DIG-labeled RNA transcripts of MAGP-1 cDNA clone
cM32 (lane 1) and MAGP-2 cDNA clone cM10A (lane 2).
The size of bovine MAGP-1 mRNA has previously been estimated as 1.1
kb(8) , and this is indicated by the arrow.
Figure 7: Restriction map of cDNA clones for bovine and human MAGP-2. The diagram shows the bovine MAGP-2 cDNA clone cM10A, identified by expression screening, compared with subsequently isolated, larger cDNAs encoding the entire amino acid sequences of bovine (cI5) and human MAGP-2 (cH61). Coding regions are shown in black and their lengths are indicated. Noncoding regions are shown in white. Polyadenylation signals (PAS) are marked.
Figure 8: Comparison of bovine and human MAGP-2 amino acid sequences deduced from cDNAs. The nucleotide sequence of bovine MAGP-2 cDNA clone cI5 is shown above the deduced amino acid sequence of bovine MAGP-2. The 5` end of clone cM10A is indicated. Peptide sequences from digests of bovine MAGP-2 are shown in italics. The human MAGP-2 sequence, translated from cDNA clone cH61, is also shown where it differs from the bovine sequence. Note the insertion of the sequence VLA after amino acid number 60. Underlined in order are: (a) an RGD putative cell-binding sequence, (b) a potential site for N-glycosylation, and (c) two AATAAA signals for polyadenylation.
Searches of the GenBank DNA and Swiss protein data bases revealed that MAGP-2 has little
similarity to other known proteins with the exception of MAGP-1. Like
MAGP-1, MAGP-2 was found to be a highly hydrophilic protein containing
two distinct domains, an acidic cysteine-free amino-terminal half and a
basic, cysteine-rich carboxyl-terminal half (Fig. 9A).
However, there are major structural differences between the proteins.
The amino-terminal domain of MAGP-2 is rich in serine and threonine
residues and lacks the proline-, glutamine-, and tyrosine-rich
sequences found in MAGP-1. This domain of MAGP-2 also contains a RGD
putative integrin-binding motif and a consensus sequence for N-glycosylation. Both motifs are lacking in the MAGP molecule.
The carboxyl-terminal domain of MAGP-2 contains 8 cysteine residues in
contrast to the 13 found in MAGP, and MAGP-2 lacks a hydrophobic region
at the extreme C terminus. Overall MAGP-2 has a relatively neutral
isoelectric point (pI 6.6), whereas MAGP-1 is highly acidic (pI 4.7).
Close sequence similarity (57%) is confined to a 60-amino acid region
in the center of the two proteins (Fig. 9B). This
region contains the first 7 cysteines of MAGP-2 and the first 8
cysteines of MAGP-1. All of these residues in MAGP-2 precisely align
with cysteines in MAGP-1, in which cysteine 3 is the unmatched residue.
The distance between each cysteine is precisely conserved between the
two proteins. The consensus sequence between the proteins is also
shown. Note that two MAGP-2 peptide sequences match those encoded by
MAGP-2 cDNA in this region and that they are clearly distinct from the
corresponding sequences of MAGP-1. It is interesting that this central
region of structural similarity between MAGP-2 and MAGP-1 corresponds
precisely, in the bovine and human MAGP-1 genes, with exons 7 and 8 and
about 20 bp of the final exon, exon 9 (27, 28) .
Figure 9: Structural comparison of MAGP-2 with MAGP-1. A, a linear representation of MAGP-2 and MAGP-1 polypeptides with their cysteine residues numbered 1-8 and 1-13, respectively. In the central region of structural similarity between the proteins, seven of the cysteines can be precisely aligned (vertical lines). Note the unmatched cysteine (number 3) in MAGP-1 which is highlighted by a star. Other structural features of MAGP-2 and MAGP-1 are also shown: vertically striped box, signal sequence; cross-hatched box, tyrosine sulfation consensus sequence; checked box, polyglutamine sequence; horizontally striped box, RGD motif; black circle, consensus sequence for attachment of N-linked carbohydrate. B, the alignment of amino acid sequences encoded by bovine MAGP-2 and MAGP-1 cDNAs in the region of close similarity, together with their consensus sequence. Also shown are sequences from MAGP-2 peptides.
The predicted size of the mature MAGP-2 polypeptide encoded by the cDNA is around 17 kDa for both the bovine and human forms of the protein. This is somewhat smaller than the apparent molecular mass of 25 kDa observed on SDS-PAGE. The size discrepancy may be due to glycosylation of the protein which has a consensus sequence for N-linked carbohydrate attachment. However, it should be noted that MAGP-1, with actual molecular mass calculated as 20 kDa, migrates on gels as a 31-kDa species even in the absence of carbohydrate side chains(8) . This anomalous migration appears to be a function of the primary structure of MAGP-1 and a similar effect may explain the difference in the observed and calculated sizes of MAGP-2. Since MAGP-2 contains eight cysteine residues it is possible that these all pair to form intramolecular disulfide bonds. However, there is evidence that all of these residues are not solely involved in intramolecular linkages. MAGP-2, like fibrillin-1 and MAGP-1, is resistant to extraction from tissue homogenates with the strong chaotrope, 6 M guanidinium chloride, but it is readily solubilized in saline if a reducing agent is included to disrupt disulfide linkages(18) . This indicates that MAGP-2 forms intermolecular disulfide bonds either with other MAGP-2 molecules or with other components of the microfibrils and thus has a structural role in association with these entities.
Figure 10: The human MAGP-2 gene is located on chromosome 12 at 12p12.3-13.1 An idiogram is shown with the total number of fluorescence signals detected on chromosome 12 from 25 metaphases using a human MAGP-2 cDNA probe. Each black circle represents an individual signal.
It is long established that the elastin-associated microfibrils are distinct in composition from elastin itself on the basis of major differences in amino acid composition, glycosylation, and susceptibility to digestion with different proteases(1) . However, it is only recently that individual glycoproteins and proteins have been identified with the microfibrils by immunoelectron microscopy. Progress has been further hampered by observations that a number of serum components including serum amyloid P, fibronectin, and vitronectin appear to become increasingly bound to the microfibrils with tissue aging(2) . However, an increasing number of proteins and glycoproteins have been immunolocalized to these structures in developing tissues. Many of these candidate microfibrillar proteins have been cloned and sequenced. These include: fibrillins 1 and 2 (350 kDa); MAGP, also recently described as MFAP2 (31 kDa); MFAP1 (57 kDa); MFAP3 (41 kDa); and the 36-kDa MFAP4(8, 9, 11, 29, 30, 31, 32) . Unlike the fibrillins, MFAPs 1-4 do not exhibit structural similarities to each other.
It is now established that the major structural elements of the microfibrils are the fibrillins, which are large rodlike glycoproteins. Evidence suggests that they are arranged as parallel bundles of 6-8 molecules joined end to end in a head to tail manner(33) . It is not yet clear if fibrillin-1 and fibrillin-2 exclusively form distinct microfibrils or if they coexist within the same microfibril in some instances(11, 12) . Rotary shadowing of microfibrils has revealed a ``beads on a string'' morphology for these structures(33, 34) . The interbead regions appear to correspond to fibrillin bundles but the composition of the beads is less clear. MAGP-1 is a small acidic, elastin-binding protein which is associated with these beadlike structures where it forms disulfide and possibly transglutaminase cross-links(7, 8, 35) . Thus MAGP-1 may play roles in stabilization of the end to end and/or lateral aggregation of fibrillin bundles within the microfibril and also in the binding and alignment of the elastin precursor, tropoelastin, to the microfibrils during elastic fiber development(2) .
We have previously described the identification of four polypeptides, which we provisionally named MP340, MP78, MP70, and MP25 (now referred to as MAGP-2), that were extracted in close association with MAGP-1 from the elastin-rich nuchal ligament of fetal calves. Affinity-purified antibodies to MP340 and a mixture of MP78 and MP70 were shown to localize to the 12-nm microfibrils in both elastic and nonelastic tissues, indicating that the proteins were associated with these structures(18) .
In order to characterize further and possibly clone the above polypeptides, peptide digests were made, and individual peptides were purified and sequenced. Several peptide sequences from MP340 were shown to match closely sequences found in fibrillin-1(10) . This confirmed earlier suggestions that MP340 is bovine fibrillin-1 (18) . It is noteworthy that none of the peptides matched fibrillin-2 sequences (11) indicating that reductive saline extracts of nuchal ligament are substantially free of this form of fibrillin. This suggests either that the two fibrillins have significantly different solubility properties or that fibrillin-1 is the predominant form of fibrillin present at this stage of development in the nuchal ligament.
Peptide mapping strongly
indicates that MP78 and MP70 are isoforms of the same protein. This may
explain the difficulties encountered in attempts to separate them
chromatographically. A close similarity was found between sequences of
these peptides and the recently identified human protein ig-h3,
inferring that MP78/70 is the bovine version of the same protein.
ig-h3 is a novel 68-kDa protein recently discovered by
differential screening of cDNAs made from A549 human lung
adenocarcinoma cells in which the expression of the protein was
stimulated by transforming growth factor-
1(20) . The
predicted 683-amino acid sequence of secreted
ig-h3 contains four
regions of internal homology, 11 cysteine residues, and a RGD motif
which may represent an integrin binding sequence(36) . More
recently, a recombinant form of the protein was shown to block the
adhesion of several cell types to plastic culture dishes, leading the
authors to suggest
ig-h3 may be an extracellular matrix
protein(37) . This anti-adhesion effect was not mediated by the
RGD motif as the carboxyl-terminal region containing the RGD sequence
was found to be absent from the recombinant protein. The evidence
suggested that the 68-kDa recombinant protein had been processed from a
larger form of
ig-h3 which has a predicted molecular weight of
76,000. It seems likely that MP78 represents the larger form and MP70
relates to the processed form of bovine
ig-h3. Interestingly, in
an independent study of human eye tissue by Escribano et
al.(38) ,
ig-h3 was found to be expressed by two cell
lines derived from ciliary epithelium. Ciliary epithelium is the tissue
which attaches the microfibril-rich ciliary zonule to the ciliary body
and thus it is possible that the epithelial cells synthesize
ig-h3
for incorporation into the zonular microfibrils. In the same study
ig-h3 was also found, by immunofluorescence, to be present on the
extracellular surface of corneal epithelial cells(38) . Our own
studies indicate that
ig-h3 (MP78/70) can become tightly bound to
the extracellular matrix, where it appears to be associated with the
microfibrillar proteins, fibrillin-1 and MAGP-1. Therefore, it seems
that
ig-h3 may be associated both with microfibrils and with the
cell surface. This behavior is reminiscent of that of the 67-kDa
elastin-binding protein, also identified in zonular
extracts(39) , which is considered to direct the secretion of
elastin from the endoplasmic reticulum to elastic fiber assembly sites
at the cell surface(40, 41) . The primary structure of
the 67-kDa elastin-binding protein is still uncertain, although recent
evidence suggests that it may be an alternatively spliced form of
-galactosidase(42) . There is an intriguing possibility
that
ig-h3/(MP78/70) may be related structurally and/or
functionally to the 67-kDa elastin-binding protein.
It is
interesting that the human gene for ig-h3 is located on chromosome
5 at band q31 (37) which is close to the loci of the
fibrillin-2 gene (5q21-31) (14) and the gene for another
41-kDa microfibril-associated protein known as MFAP3
(5q32-33.2)(31) . The loci of fibrillin-1 and MFAP-1
genes are also close together on chromosome
15(14, 30, 43) . It is possible that this
gene clustering serves some purpose in the coordination of gene
expression of microfibrillar proteins. However, it should be noted that
the genes for several collagens and fibronectin, which are not
necessarily coordinately expressed with each other, are located in
close proximity on chromosome 2(44) .
Analysis of peptide
sequences from MAGP-2 indicated that the protein was distinct from all
known proteins. Affinity-purified anti-MAGP-2 antibodies
immunolocalized strongly to the elastin-associated microfibrils but
showed no cross-reactivity with fibrillin, ig-h3, or MAGP-1. This
confirmed that MAGP-2 was also a discrete microfibrillar component.
Molecular cloning was used to obtain the entire coding sequences of its
bovine and human genes. Interestingly, data base searches revealed
little sequence similarity to other known proteins with the exception
of MAGP-1(8) . MAGP-1 and MAGP-2 were found to have an
extensive region of sequence resemblance, and thus they appear to
represent a new family of microfibril-associated proteins. In
acknowledgment of their structural similarity, we support and retain
the names MAGP-1 and MAGP-2 in preference to MFAP-2 and MFAP5. Neither
protein has a structural relationship to MFAP-1, MFAP-3, or MFAP-4.
While the close resemblance of the central regions of MAGP-2 and MAGP-1 suggests that the two molecules may share some activities, the structural diversity of other regions indicates that the proteins may also have distinct functions. MAGP-2 lacks tyrosine-rich and glutamine-rich motifs in the amino-terminal region and a hydrophobic carboxyl-terminal region, found in MAGP-1(8) . These domains may be important for the known modifications and interactions of MAGP-1 which include tyrosine sulfation, transglutamine cross-linking, and tropoelastin binding(35) . It will be interesting to establish if any of these properties are shared with MAGP-2. Unlike MAGP-2, MAGP-1 contains an odd number of cysteine residues, and the evidence presented here suggests that cysteine 3 is the unpaired cysteine. This residue is likely to confer on MAGP-1 some additional intermolecular disulfide bonding properties. In contrast, the presence of a conserved RGD motif in a hydrophilic sequence of MAGP-2 suggests that it may have integrin-binding properties, lacking in MAGP-1, and thus may play an additional role in the interaction of the microfibrils with the cell surface.
Preliminary studies on the distribution of MAGP-2 during
tissue development indicate that the protein is associated with
elastin-associated microfibrils, and with elastin-free microfibrils in
a number of tissues. However, MAGP-2 appears to have a more restricted
tissue distribution than MAGP-1 and fibrillin-1 in late-fetal and adult
tissues. For instance, our studies suggest that MAGP-2 is absent from
the microfibrils of the ciliary zonule and the connective tissue around
kidney tubules. Also MAGP-2 gene expression appears to be more closely
linked than that of MAGP-1 to the switching on of the elastin gene in
developing nuchal ligament. ()These findings suggest that
MAGP-2 may be involved in developmental and tissue-specific modulation
of microfibril function. Interestingly, a recent study has shown that,
during murine embryogenesis, fibrillin-2 has a more restricted profile
of expression than fibrillin-1 in terms of both developmental stages
and tissue distribution(12) . Fibrillin-2 generally appeared
earlier and accumulated for a shorter period than fibrillin-1. Of
particular note are the findings that the expression of fibrillin-2 is
very low or absent from the ciliary zonule and that, in kidney, it is
restricted to the glomerulus(12) . This indicates that there
are similarities in the tissue distributions of MAGP-2 and fibrillin-2
and raises the possibility that MAGP-2 is specifically associated with
fibrillin-2-containing microfibrils. To test this idea we are
conducting a more thorough study of MAGP-2 expression patterns. In
addition, further structural characterization of MAGP-2 and its human
gene, which we have located on chromosome 12 at 12p12.3-12p13.1,
is in progress.
In conclusion, molecular cloning and peptide sequencing techniques have been instrumental in determining the structural inter-relationships of five polypeptides which we have identified with the microfibrillar component of elastic fibers. The polypeptides are derived from four distinct proteins, two of which are structurally related to each other. A major challenge is ahead to determine how these microfibril-associated proteins interact with one another and with other proteins. This in turn will lead to a better understanding of the function of microfibrils, in elastic and nonelastic tissues, including the role of these structures in the complex process of elastic fiber assembly.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Bos taurus U37282[GenBank], Homo sapiens U37283[GenBank].