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Papers of the Month - August 1996
It's been a *busy* month, so I'm going to take a shortcut. A brief
paper from my lab was just published in Biochem Biophys Research
Communications, 1996; 225: 500-504 [August 14]. I'm going to give
you the text, minus Table I and the References (which I can't get to
format properly with my software). I think we have stumbled onto a
whole new class of human genes and proteins. Our present speculation
is that the kappafibs may be interacting with the integrins, since
integrins use immunoglobulin motifs in their cell-cell interactions.
So why shouldn't the integrins interact with Ig motifs in the matrix ?!
TWO HYPOTHETICAL PROTEINS OF HUMAN AORTIC
ADVENTITIA, WITH Ig KAPPA, COLLAGENOUS,
AND AROMATIC-RICH MOTIFS
Kathleen J. Ozsvath, Shichao Xia, Hitoshi Hirose,
and M. David Tilson
From the Department of Surgery, College of Physicians
and Surgeons, Columbia University, New York, New York
-----------------------------------------------
Summary: Two of five clones, selected from an
expression library of aortic adventitia, encode unique
hypothetical proteins sharing sequences of Ig kappa,
gly/pro rich (collagenous) motifs, and aromatic motifs
that occur in several proteins of the extracellular
matrix. Both proteins have a similar domain structure
with at least 8 regions: 1) Ig kappa (84-120 residues
in length); 2) ser/thr-rich motif (44-63); 3) a second
Ig kappa motif (9-12); 4) either a possible calcium-
binding motif or a gly/pro rich sequence (35-43); 5) an
aromatic rich sequence (6-7); 6) another gly/pro rich
sequence (62-72); 7) a third Ig kappa sequence (26-28);
and 8) a C-terminal 68-70 residue sequence with another
aromatic motif.
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-
We have reported the partial amino acid sequence
of a human microfibrillar glycoprotein ~40 kDa, which
we called Aortic Aneurysm-associated Antigenic Protein-
40 kDa (AAAP-40).() It has sequence homologies with
vitronectin (VN) and fibrinogen (FB). A cDNA library
was prepared from mRNA purified from human aortic
adventitia. Since VN and FB are not synthesized in
aorta, the library was screened with polyclonal
antibodies to VN and FB. The hypothetical proteins of
clones 1 and 5 share a novel domain structure and are
the subject of this communication.
Methods
mRNA from aortic adventitia was reverse transcribed
and inserted into the phagemid, Uni Zap XRTM lambda, by
arrangement with Stratagene (La Jolla, CA). The phagemid
was transfected into E Coli (XL1-Blue MRF|, StratageneTM).
The cells were plated on top agar and grown at 420C until
small plaques were visible. Nitrocellulose membranes
impregnanted with 10 mM isopropyl thio-beta-D-
galactopyranoside were placed on the agar and incubated
for 6 hours at 37oC. The membranes were removed and
blocked with 5% milk in TBS for 45 minutes. Incubation
was continued with either rabbit anti-human vitronectin
(SigmaTM,St. Louis, MO) (1:10,000) or fibrinogen antibody
(SigmaTM 1:2,500) for 2 hours at room temperature. The
membranes were washed in TBS and incubated with alkaline-
phosphatase-conjugated goat anti-rabbit IgG (1:5000)
(SigmaTM) for 2 hours at room temperature. Following a
series of washes in TBS, the membranes were developed by
the Vectastain NBT/BICP color reagent system. The
positive plaques were rescreened to obtain pure clones.
Excision from positive clones was carried out using
the Ex Assist/SOLR System (Strategene TM). The phages
were extracted and re-transfected to SOLR cells with
amplified Ex Assist Helper Phage (Strategene TM). These
cells were grown overnight at 37o C, on ampicillin-
supplemented medium, which inhibits the growth of non-
transfected cells. The cells were then harvested and
lysed by alkali buffer, and the DNA was purified by
phenol-chloroform extraction. DNA sequencing was carried
out by the core laboratories at Columbia College of
Physicians and Surgeons, Columbia University, NY, NY.
Immunohistochemistry was performed with rabbit anti-
human Ig kappa, 1:50, (SigmaTM) and APC goat anti-rabbit
IgG, 1:50, whole molecule, (SigmaTM). APC goat anti-
human Ig heavy chain (FC specific, SigmaTM), 1:150, was
used for control experiments. The slides were developed
with BCIP-NBT kit (Biomeda, Foster City, CA) and
counterstained with Fast Red (SigmaTM).
Results
The hypothetical proteins encoded by clones 1 and
5 have similar features and domain structures, which
can be aligned in eight regions beginning with the N-
terminus (Tables I and II). Region 1 is a lengthy
sequence highly homologous to Ig kappa V (88-120
residues). Region 2 is a 44-63 residue sequence, which
in the case of clone 5 has a six residue sequence that
also occurs in cytomegalovirus. Region 3 is a 9-12
residue sequence that is conserved from Ig-kappa.
Region 4 is a possible calcium-binding motif in clone
1, while clone 5 has a gly/pro rich sequence. Region 5
is an aromatic-rich sequence. Region 6 is a gly/pro
rich sequence in both clones. Region 7 returns to a
conserved sequence from Ig kappa, which in clone 1
contains an RGE motif. Region 8 is a C-terminal
sequence of 68-70 residues containing a second aromatic
motif.
Immunohistochemistry revealed binding of rabbit
anti-human Ig kappa antibody to the aortic adventital
microfibril, in specimens of both normal and aneurysmal
human abdominal aorta.
Discussion
These are novel hypothetical proteins. The use of
an immunoglobulin domain as a specificity determinant
has been described in the fibroblast growth factor
receptor,() but the use of Ig kappa sequences in
matrix proteins has not been described. The gly/pro
rich sequences are typical of the collagens. The
aromatic motifs resemble similar motifs in vitronectin
(VN: FFFS), microfibril-associated protein 4 ()
(MFAP-4: FYYS), and AAAP-40 (FFYS and YY.FFQYT).(1)
GenBank searches have led to the conclusion that
aromatic motifs, followed by SP, TS, or L, are rare in
structural mammalian proteins, except for the above-
mentioned. FFFSP occurs in cytomegalovirus (CMV)(),
as does the sequence CRIKN.AV in clone 5, consistent
with the hypothesis that the clinical association of
aneurysm disease and infection with CMV () may be on
the basis of molecular mimicry.() FFFL is limited to
a human T-cell receptor, a secretory product in mouse,
and herpes virus 2 @ residue 62. The herpes virus is
interesting, because it is another potential molecular
mimic.(6, ) FFTS is reported only in one other
protein, lens fiber major protein.() Another
interesting feature of clone 1 is the possible calcium-
binding sequence. The importance of calcium in the
self-assembly of the microfibril has been
documented,() and we have previously reported a
possible calcium-binding motif in AAAP-40.(1) MAGP-36
is also a calcium-binding microfibrillar protein, with
tissue distribution limited to the aorta in pig.()
Because of these unique features, we suggest the
name "kappafibs" for these hypothetical proteins, to
reflect their use of Ig kappa motifs and their possible
role as structural elements of the microfibril. Table I - Clone 1. Amino acid sequence of the
hypothetical protein encoded by clone 1. "*" = cystein
residue used in disulfide bond of Ig kappa, present
(along with the following R.SQ) in both clones. "X" =
indeterminant residue.
Table II - Clone 5. Amino acid sequence of hypothetical
protein of clone 5.
*
MVLQTQVFISLLLWISGANGDIVMTQSPDSLGVSLGERATINCRSSQRL 49 Clone 5
MVLQTQVFISLLLWISGA+GDIVMTQSPDSLAVSLGERATINCKSSQSV IgK V-IV ()
<<--First Igk homology region begins at #1-------
LL.L.V DSL V.L MFAP-4
FFGSNSKNYLAWYQQKPGQSPKLLIYWASTRDSGVLTDSLAAGLGXI 96 Clone 5
LYSSNNKNYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTD Ig kappa
----------First Igk motif ends---->
SLSPSXXCRLKNLAILXLSAIIIISXXTFRPWGTXLXIQXKCWXAXIFXSF 147 - Clone 5
FTLTISRLEPEDFAVYYGQQYGSSPLTFGGGTKVEIKRTVAA----PSVFI Ig kappa
SISP MAGP-36
SISP LLLLS MFAP-4
CRIKN.AV Cytomeg
FPPXEKQFK-<----First gly/pro rich motif()------> 218 Clone 5
FPPSDEQLK---------(joins EI below)---------------- Ig kappa
<2nd Igk
Motif>
FFFFSPFLXGWXLGXLFXGPXEKIFFPXGPKKRGRGXKXPPNWGKSPSG 268 Clone5
------------------EITASVVGLLNNFYPREAKVQWKVDNALQSG Ig kappa
motif>
FFFSP Cytomegaly V
FYYS AAAP-40
FYYS MFAP-4
FFFS Vitronectin
FFF.PF Clone 1
XXXGRGXQGKGNLKALWXEPXRLGKGGIRGXNKXXAXEVTHSGLSFAXSKKXXQGRX Clone 5
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAGEVTHQGLSSPVTKSFNRGEG Ig k
-G/P motif <3rd Igk Homology region>
ends ------> A.AA...K.KVYA.EVTH.GL....S....E Clone 1
LEGEVPPPVXXXQPDPLPSFGLXPFFHRGXTPIXVXQXIFYXTPLXXLGFNYXNVXXXXINKVXFL
#393
LEGEND FOR FIGURE
Photomicrographs of immunmohistochemical preparations
of aneurysmal (AAA) and normal (NL) aortic adventitia,
illustrating binding of rabbit anti-human Ig kappa to
the elastin-associated microfibril. Control experiments
with rabbit anti-human Ig heavy chain as first antibody
revealed minimal, if any, immunoreactivity of the micro-
fibril.
References