From the
Tubulointerstitial nephritis antigen (TIN-ag) is a 58-kDa
basement membrane glycoprotein that is recognized by human
autoantibodies in certain forms of tubulointerstitial nephritis. To
further characterize this macromolecule and isolate cDNAs encoding
TIN-ag, amino acid sequences from tryptic peptides were used to design
and synthesize primers in order to amplify a probe for screening a
rabbit kidney cortex cDNA library. A cDNA encoding TIN-ag was cloned
and sequenced. The predicted amino acid sequence deduced from this cDNA
includes the chemically determined sequences of peptides derived from
TIN-ag, supporting its authenticity. The predicted amino acid sequence
also shows that the carboxyl-terminal region of the molecule exhibits a
30% homology with human preprocathepsin B, a member of the cysteine
proteinase family of proteins. A domain in the amino-terminal region of
TIN-ag contains an epidermal growth factor-like motif that shares
homology with laminin A and S chains,
Immunologically mediated tubulointerstitial nephritis
(TIN)
This laboratory has focused on the identification and
characterization of a 58-kDa novel basement membrane protein,
tubulointerstitial nephritis antigen (TIN-ag), previously reported to
be associated with anti-TBM TIN(9) . This macromolecule was
eventually purified from rabbit TBM. Limited amino acid sequencing
indicated that TIN-ag is a novel component of the basement
membrane(10) .
Immunofluorescence studies have revealed an
interesting pattern of reactivity(9) . TIN-ag was detected
primarily in the basement membrane underlying proximal tubular
epithelial cells, to a lesser extent in Bowman's capsule and the
basement membrane of the distal tubules, and was absent from the
glomerular basement membrane and the mesangial matrix. In extrarenal
tissues, TIN-ag was detected in part of the small intestine (ileum) and
the corneal and epidermal basement membranes.
Recently, in vitro solid phase binding studies revealed that TIN-ag reacted with both
type IV collagen and laminin but not with heparin(11) . These
reactions were found to be specific and saturable with an affinity in
the micromolar range. Furthermore, TIN-ag exhibited a
concentration-dependent inhibitory effect on the polymerization of
laminin and on preformed laminin networks. This observation is of
importance in view of the fact that laminin polymers have been shown to
exist in vivo, and therefore the presence of TIN-ag in certain
areas may profoundly effect the structure of the specific basement
membranes.
A fundamental understanding of the molecular properties
of this potential nephritogenic antigen is essential to understand the
normal physiological contribution of these macromolecules, and in
delineating the pathophysiology of interstitial nephritis associated
with basement membrane macromolecules. In this investigation the cDNA
encoding TIN-ag was cloned from a rabbit kidney cortex library. The
predicted amino acid se-quence of TIN-ag is presented and compared with
other known sequences.
To facilitate the isolation of cDNA sequences encoding rabbit
TIN-ag, we took advantage of the known amino acid sequence of the amino
terminus and an internal tryptic peptide(10) . To amplify a
specific probe, degenerate primers corresponding to the known TIN-ag
amino terminus (5` WSY ATA TTY CAR GGI CAR TA 3`) and an internally
derived amino acid sequence from the previously described TIN-ag
tryptic T4-13 peptide (3` GGI CTY TGI TGI CTR RAI GGI CT 5`) were
used to amplify a cDNA from oligo(dt)-primed rabbit kidney total RNA (Fig. 1A). The reaction amplified a fragment of about
500 base pairs and required both the amino-terminal primer and the
T4-13 primer. The 500-base pair amplified product was
gel-purified and cloned into Novagen PT7 Blue vector. Preliminary
double-stranded sequence analysis using 5` U-19 mer primer in
association with vector-specific T7 primer revealed no homology with
any known sequences in GenBank
The deduced amino acid
sequence contains seven potential glycosylation sites
(Asn-X-Ser/Thr)(20) , shown in Fig. 2. Cysteine
residues are clustered in two discrete regions: amino acids
55-152 and amino acids 238-349. No RGD sequence is
indicated in the predicted amino acid sequence of TIN-ag. A relatively
high content (3.0%) of tryptophan is also observed.
In this report we provide the full coding sequence of the
TIN-ag cDNA. The cDNA encodes a protein with a predicted molecular
weight of 54.5 kDa that shares 30% homology with the cysteine
proteinase family of molecules. Several chemically determined tryptic
peptide sequences were present within the deduced amino acid sequence,
providing evidence that the cDNA corresponds to the protein originally
isolated from TBM. Southern blot analysis of genomic DNA reveals the
presence of gene sequences that correspond to the isolated cDNA. A
full-length construct derived from the isolated cDNAs was used to
generate a recombinant protein. Immunoprecipitation of the in vitro generated recombinant product supports the hypothesis that the
TIN-ag cDNA encodes a product associated with anti-TBM TIN. The
structural data provided in this report, in combination with previously
reported functional characteristics of TIN-ag suggest that TIN-ag may
represent a macromolecule of importance in renal basement membrane
biology.
Collectively, the results of our investigation are
suggestive of TIN-ag being a member of two distinct protein
superfamilies(23) . The NH
The identification of TBM components reactive with
antisera from patients with anti-tubular basement membrane nephritis
has yielded much information regarding the heterogeneous nature of this
biologically active barrier. Clayman et al.(4) identified a 48-kDa nephritogenic antigen, 3M-1,
isolated from rabbit tubular basement membrane capable of inducing
anti-TBM disease in rodents. Nielson et al.(5) further
characterized the human 3M-1 glycoprotein and defined it as a major
nephritogenic antigen in anti-TBM disease. Yoshioka (8) has also
described nephritogenic antigens of 54 and 48 kDa isolated from human
TBM through immunoprecipitation with human patient antisera.
Interestingly, the same patient antisera used to characterize TIN-ag is
also capable of immunoprecipitating the 3M-1 glycoprotein described by
Clayman et al.(4) and Nielson et al.(5) as well as the nephritogenic antigens reported by
Yoshioka et al.(8) . This observation suggests the
presence of multiple anti-TBM antibodies present in the patient
antisera, but it also may indicate that the antigens have similar
nephritogenic domains, are structurally related, or are conferred
similar post-translational modifications. This latter interpretation is
not entirely consistent with the data presented on the characterization
of the 3M-1 glycoprotein by Neilson et al.(5) , since
peptides thought to represent the major nephritogenic domain of 3M-1
show no similarities to the predicted amino acid sequence of our
TIN-ag. Furthermore, biochemical analysis of 3M-1 reveals little
similarity to TIN-ag in amino acid composition. These two observations
suggest that the two antigens differ structurally and biochemically.
Although immunofluorescence data using monoclonal antibodies against
the nephritogenic antigen described by Yoshioka etal.(8) show similar tissue distribution as TIN-ag, the
reported NH
Basement membranes represent highly specialized barriers that are
present between the basal surface of polarized cells and the
interstitial tissue. They are thought to contribute significantly to
cellular phenotype, vascular permeability, and overall tissue
architecture. Their macromolecular constituents are involved in these
physiological processess. Knowledge of the TIN-ag amino acid sequence
may provide valuable insight into the possible physiological role of
this macromolecule in normal tissue and facilitate an understanding of
diseases that are associated with the basement membrane. Furthermore,
because the highest degree of immunohistochemical staining for TIN-ag
is in the basement membranes beneath epithelial cells involved in the
active transport of many metabolites including proteins, peptide
molecules, and electrolytes, it is tempting to speculate that TIN-ag
plays a crucial role in the creation and maintenance of these
physiological environments.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
chain of type I
collagen, von Willebrand's factor, and mucin, suggesting
structural and perhaps functional similarities among these molecules.
Immunoprecipitation of in vitro generated recombinant protein
using a TIN-ag-specific monoclonal antibody (A8), confirms the identity
of the isolated TIN-ag cDNA. In this report the cDNA and predicted
amino acid sequences of TIN-ag are presented. Knowledge of the primary
structure of TIN-ag will facilitate our understanding of the molecular
structure of this novel basement membrane component and may provide
clues toward understanding its functional role.
(
)is characterized by the linear deposition
of immunogammaglobulin (IgG) and complement along the renal tubular
basement membrane (TBM). Antibodies to the TBM have been identified in
patients with TIN, various types of glomerular nephropathies, and
recipients of a renal allograft(1) . Tubulointerstitial
nephritis may be either acute or chronic. Acute tubulointerstitial
nephritis is characterized by the presence of interstitial edema,
leukocytic infiltrates, and patchy tubular necrosis, whereas chronic
tubulointerstitial nephritis is characterized by a mononuclear
infiltration accompanied by widespread tubular fibrosis and extensive
tubular atrophy. Alterations of the TBM as a result of the accumulation
of anti-tubular basement membrane antibodies and complement may
contribute to altered renal function and may eventually lead to end
stage renal disease(2) . The molecular origin of the tubular
basement membrane macromolecule(s) that are reactive with anti-tubular
basement membrane antibodies in patients with tubulointerstitial
nephritis has been the subject of many investigations. Immunologically
mediated forms of TIN have been described in various animal models
typically induced following immunization with kidney TBM. These studies
have led to the description of various nephritogenic antigens between
30 and 70
kDa(3, 4, 5, 6, 7, 8) .
Cloning of TIN-ag cDNA
A rabbit kidney cortex
library cloned into gt11 was screened for cDNAs encoding TIN-ag. A
500-base pair TIN-ag cDNA clone was polymerase chain reaction-amplified
using degenerate primer sequences based upon previously reported
tryptic peptides (10) from oligo(dt)-primed rabbit kidney total
RNA. The conditions for amplification were as follows: 94 °C for 5
min; 37 °C for 2 min (two cycles) followed by 35 cycles at 72
°C for 3 min; 94 °C for 1 min 20 s; 37 °C for 2 min; and
finally at 72 °C for 7 min. This amplified fragment was sequenced,
subsequently random prime-labeled with
[
-
P]dCTP, and was then used to screen
duplicate nitrocellulose filter lifts under high stringency conditions
(6
SSC, 0.5% SDS twice at room temperature for 30 min each; 0.1
SSC, 0.1% SDS for 60 min at 65 °C). Five positive clones of
various lengths were identified and subcloned into Stratagene
pBluescript II SK
EcoRI site. DNA sequencing
of the putative TIN-ag clones was performed by the dideoxynucleotide
chain termination method of Sanger(12) , using Life Sciences
Sequenase version 2.0. Both strands of the TIN-ag final construct were
sequenced using a combination of vector-specific primers (T3 and T7)
and internally derived sequence-specific primers. When
sequence-specific primers were employed, all junctional sequences were
included in sequence analysis.
TIN-ag Sequence Homology Searches
The nucleotide
sequence and predicted amino acid sequence of TIN-ag were subjected to
a data base search against the GenBank nucleotide and protein sequence
data bases (release 72.0). Alignment of TIN-ag predicted amino acid
sequence was performed by DNASTAR version 2.0 software utilizing the
Pearson and Lipman (13) method of sequence alignment to
determine the regions of maximum homology.
Southern Blot Hybridization
Ten µg of rabbit
genomic DNA was digested at 37 °C for 4 h with restriction enzymes
HIND III and EcoRI for Southern analysis(14) . The
samples were electrophoresed through a .8% agarose Tris Borate EDTA
gel. Following electrophoresis, the gel was depurinated (5 min in .25 M HCl), rinsed with denaturation buffer (0.5 M NaOH,
1.5 M NaCl), and blotted onto a nylon membrane (Boehringer
Mannheim) overnight in denaturation buffer. The membrane was
cross-linked using a stratalinker (Stratagene) prehybridized at 42
°C for 4 h, and then probed using an SphI/EcoRI
cDNA fragment of TIN-ag, which was random primed with
[P]dCTP according to the manufacturer's
protocol (Boehringer Mannheim random prime labeling kit for nucleic
acids). The blot was washed for 30 min in 6
SSC, 0.5% SDS for
30 min at room temperature and finally at 62 °C in 0.1
SSC,
0.1% SDS for 1 h. The Southern membrane was then exposed to x-ray film
at -70 °C for 48 h using an intensifying screen.
In Vitro Transcription of TIN-ag cDNA
The
full-length TIN-ag construct was subcloned into pBluescript II
SK and transcribed using vector-specific RNA
polymerase T7. Two micrograms of nonlinearized TIN-ag plasmid DNA was
purified using the GeneClean kit (BIO 101, Inc.) and resuspended in 2
µl of diethyl pyrocarbonate-treated H
O. To this, 5
µl of transcription buffer (200 mM Tris-HCl (pH 8.0), 40
mM MgCl
, 10 mM spermidine, 250 mM NaCl) was added to the template. Next, 1.0 µl each of 10
mM rATP, rGTP, rCTP, rUTP, and 0.75 M dithiothreitol
was added in addition to 40 units of RNasin RNase inhibitor (Promega)
to the transcription reaction. After the addition of 10 units of T7 RNA
polymerase, diethyl pyrocarbonate H
O water was added to a
final volume of 25 µl. The reaction was incubated in a 37 °C
water bath for 30 min. DNA templates present in the transcription
reaction were removed by incubation at 37 °C with 10 units of
RNase-free DNase/µg of DNA template for 15 min. TIN-ag RNA was
extracted once with phenol:chloroform (v/v) (1:1) and precipitated with
0.1 volumes of 3 M NaOAc (pH 5.2) and 3 volumes of ethanol.
The mRNA was centrifuged at 14,000
g for 15 min; the
RNA pellet was allowed to dry, and was resuspended in 3 µl of
diethyl pyrocarbonate-treated H
O.
In Vitro Translation of TIN-ag mRNA
The mRNA from
the above transcription reaction was heated to 68 °C for 1 min to
eliminate any secondary structure formation in the mRNA. Two µl of
nonradioactive amino acid mixture was added to the reaction to a final
concentration of 50 µM. Twenty µl of rabbit
reticulocyte lysate (Stratagene) was added, and after thorough mixing
was allowed to proceed for 60 min at 30 °C. Following the
translation reaction, the contents of the tube were centrifuged and
stored at -20 °C.
Electrophoresis Techniques
SDS-polyacrylamide gel
electrophoresis was performed as described by Laemmli (15) on
10% gels. Gels were run for 4-5 h at approximately 30 mA.
Immunochemical Techniques
Protein A-agarose was
used to immunoprecipitate the immune complexes formed at 4 °C from
a lysate containing the in vitro translated product from the
TIN-ag cDNA clone. Rabbit anti-mouse IgG was used as a bridging
molecule between the protein A-agarose and the primary antibody. The
following general procedure was carried out as follows in a 1.5-ml
conical microfuge tube. Protein A-agarose beads were conjugated to
rabbit anti-mouse IgG by incubation at room temperature for 1 h.
Following this, the conjugated complex was washed 3 times in
immunoprecipitation buffer (IP buffer) (50 mM Tris-HCl, 0.14 M NaCl, 1.0% Triton X-100, 1 mM CaCl, 1
mM MgCl
, 1 mM MnCl
, 1 mM phenylmethylsulfonyl fluoride, 1 mMN-ethylmaleimide, 0.2% NaN
, pH 7.2). Normal
mouse IgG was then conjugated to the complex, and the reaction was
placed on a rotary platform at room temperature for 1 h. Following this
incubation, the tubes were washed 3 times with IP buffer, and the
samples were added to the tubes for preclearance of nonspecific binding
proteins at 4 °C overnight. Samples were washed 3 times with IP
buffer, and supernatants were transferred to tubes containing the
primary antibody (A8), which was raised against purified TIN-ag
isolated from tubular basement membrane. This complex was conjugated to
rabbit anti-mouse and protein A-agarose beads for immunoprecipitation
of the desired in vitro expression product of TIN-ag at 4
°C overnight. Samples were washed 4 times in IP wash buffer (50
mM Tris-HCl, 0.4 M NaCl, 1.0% Triton X-100, 1 mM CaCl
, 1 mM MgCl
, 1 mM MnCl
, 1 mM phenylmethylsulfonyl fluoride, 1
mMN-ethylmaleimide, 0.2% NaN
, pH 7.2)
and centrifuged to collect pellet.
Western Blotting
Immunoprecipitation samples were
resuspended in loading buffer and subjected to SDS-polyacrylamide gel
electrophoresis in 10% polyacrylamide gels according to the methods of
Laemmli et al.(15) . Protein was then
electrophoretically transferred (0.2 A for 2 h) from the polyacrylamide
gel to nylon membranes (Immobilon-P; Millipore, Bedford, MA) as
described by Towbin et al.(16) , and unoccupied sites
on the nitrocellulose were blocked by incubating with PBS containing 3%
skim milk overnight at 4.0 °C. The blocked membrane was incubated
for 1 h at room temperature with a previously described TIN-ag
monoclonal antibody (A8) diluted 1:50 in a solution of PBS and 3% skim
milk. The membrane was then washed with PBS 4 times for 15 min and
incubated for 30 min at room temperature with horseradish
peroxidase-conjugated goat anti-mouse IgG antibody (UBI, Lake Placid,
NY) at a 1:2000 dilution in PBS containing 0.05% Tween 20 (TPBS). After
washing the membrane 2 times for 15 min and 4 times for 5 min each with
TPBS, the bound secondary antibody was detected by enhanced
chemiluminescence (Amersham Corp.) according to the
manufacturer's protocol.
. Sequence data indicated
the presence of both primer sequences, and the amino acid sequence of
the regions adjacent to the primers corresponded to the previously
determined peptide sequence, supporting the notion that sequences
encoding the tryptic fragment have been isolated in the cDNA clone. The
amplified product was used to screen a
gt11 rabbit kidney cortex
library for additional clones. Five positive clones of various lengths
were identified and subcloned into Stratagene pBluescript II
SK
EcoRI site for further sequence analysis.
A consensus sequence for both strands of the full-length TIN-ag final
construct was determined using a combination of vector-specific primers
(T3 and T7) and internally derived sequence-specific primers (Fig. 1B).
Figure 1:
A, location of peptides used in the
design of a specific TIN-ag cDNA probe. The locations of the peptides
(NH peptide and T4-13 peptide) that were used to
amplify a 500-base pair cDNA from an oligo(dt)-primed rabbit kidney
cortex library are indicated by arrows. The amplimer generated
in the above reaction was used to screen a
gt11 rabbit kidney
cortex library for additional TIN-ag cDNA clones. B, TIN-AG
sequencing strategy. Arrows indicate the primer positions used
in the sequencing of the TIN-ag cDNA clone. Both strands of the TIN-ag
cDNA clone were sequenced using a combination of vector-specific (T7
and T3) and internally derived primers. Junctional sequences were
confirmed. Arrows indicate 5` to 3`
direction.
Sequence analysis indicated the presence
of a 1425-base pair open reading frame encoding a protein of 474 amino
acids. The 5`-untranslated region of the cDNA contains 120 nucleotides.
The 3`-untranslated region contains a canonical hexanucleotide
polyadenylation signal located 200 nucleotides downstream of the stop
codon(17) . The originally described tryptic peptide fragments
are present in the deduced amino acid sequence, providing evidence that
the cDNA that was cloned from the rabbit kidney cortex library
corresponds to the protein species initially isolated from rabbit
tubular basement membrane. The amino-terminal region of the protein
(amino acids 1-18), containing a charged NH-terminal,
central hydrophobic, and polar COOH-terminal region, represents the
putative signal peptide of the molecule(18) . This observed
signal sequence is characteristic for molecules associated with the
extracellular/secretory pathway. Twenty-nine amino acid residues are
present between the observed signal peptide and the previously
described NH
-terminal region (10). It is possible that this
region may represent a propeptide that is cleaved during the early
stages following protein synthesis(19) . Alternatively,
isolation procedures may have contributed to the shortening of the
amino-terminal portion of the molecule.
Figure 2:
Nucleotide and predicted amino acid
sequence of TIN-ag cDNA. Numbers indicate the position of the
first nucleotide or amino acid in each line. The tryptic
peptides (amino-terminal, T4-13, and T4-17) from which
primers were designed and used for screening of a rabbit kidney cortex
library are underlined. The putative signal peptide is shown
in brackets. The polyadenylation signal and 5`- and
3`-untranslated regions are indicated. Seven potential N-linked glycosylation sites are indicated by arrowheads.
A search for
homologies to other known proteins revealed that TIN-ag shares sequence
homology primarily with the cathepsin family of cysteine proteinases. Fig. 3compares the carboxyl region of the TIN-ag sequence with
human preprocathepsin B (HCB). Within this homologous region,
positions 366-375 of TIN-ag correspond to a previously determined
tryptic peptide sequence (T4-17) not used in primer design. The
result provides additional supporting evidence that the cDNA represents
the originally described TIN-ag. Alignment of conserved cysteines
between the molecules is suggestive of structural and perhaps
functional similarities between TIN-ag and the cathepsin family of
cysteine proteinases(21) . It is known that three distantly
located domains contribute to the conformation of the active site of
cysteine proteinases(22) . In two out of the three domains
TIN-ag contains sequences that are identical to those of the human
preprocathepsin B molecule, as well as all the other members of the
cysteine proteinase family. In the third region, a cysteine residue
considered important for the catalytic activity and present in all
members of the cysteine proteinase family is substituted by a serine
residue in TIN-ag at position 241.
Figure 3:
Alignment of amino acid sequences of human
cathepsin B and the predicted amino acid sequence of TIN-ag. Alignment
between the TIN-ag predicted amino acid sequence and human
preprocathepsin B (HCB). Gaps are indicated by dashes. Conserved residues are indicated by one-letter
codes. The two amino acids thought to constitute the active site
residues of cysteine proteinases are indicated by asterisks. Those
residues that are thought to contribute to the hydrophobic core of the
cysteine proteinase molecules are indicated by arrowheads (21).
Analysis of a domain in the amino
terminus of the molecule reveals an epidermal growth factor-like repeat
that is found within several classes of extracellular matrix molecules.
As shown in Fig. 4, alignment of extracellular matrix molecules
such as laminin A and S chains, von Willebrand's factor, mucin,
and the chain of type I collagen with that of amino
acids 118-152 of TIN-ag, revealed a highly conserved domain with
respect to the position of cysteines, again suggesting putative
structural similarities between this domain and other domains of
various extracellular matrix molecules.
Figure 4:
Alignment of TIN-ag predicted amino acid
sequence with various extracellular matrix molecules. The alignment of
conserved amino acids is suggestive of structural and functional
similarities between this region of the TIN-ag molecule and those
shown. VWF, von Willebrand's factor; LM-A,
laminin A chain; LM-S, laminin S chain; COL 1,
type I collagen
1 chain.
To verify the presence of
TIN-ag gene sequences corresponding to the isolated cDNA sequence,
Southern blot analysis was conducted. Rabbit genomic DNA, digested with
the restriction enzymes HindIII (lane1) and EcoRI (lane2) revealed a simple
endonuclease restriction fragment pattern indicative of a single gene (Fig. 5). These results demonstrate the presence of gene
sequences that correspond to the isolated cDNA sequence.
Figure 5:
Southern analysis of rabbit genomic DNA.
Ten µg of rabbit genomic DNA was digested at 37 °C for 4 h with
two restriction enzymes (HindIII (lane1)
and EcoRI (lane 2)) electrophoresed through a 0.8%
agarose gel and transferred to a nylon membrane for hybridization
analysis with an SphI, EcoRI fragment of TIN-ag. The
restriction enzymes EcoR-I and HindIII are not
predicted to cut within the TIN-ag coding
sequence.
In
vitro transcription and translation of the TIN-ag cDNA was carried
out to verify the identity of the gene product encoded by the isolated
TIN-ag cDNA. The in vitro translated product was
immunoprecipitated with monoclonal antibody A8, (Fig. 6).
Electrophoresis, under nonreducing conditions, of immunoprecipitated
protein samples revealed a band with a mobility of 52 kDa only in the
lane in which the in vitro reaction was carried out (lane3). The electrophoretic mobility of the recombinant
product is consistent with the predicted molecular weight of the
translated construct. The observed difference in the electrophoretic
mobility between the tissue-isolated TIN-ag and the recombinant product
may be attributed to various factors such as lack of carbohydrate
additions and differential disulfide bond formation. The
immunoprecipitation data provided evidence that the recombinant product
encodes the protein species that was originally characterized as being
associated with anti-TBM TIN.
Figure 6:
Immunoprecipitation of in vitro translation of TIN-ag cDNA construct. Samples from the
immunoprecipitation were analyzed by SDS-polyacrylamide gel
electrophoresis 10% gel in discontinuous buffers as described by
Laemmli (15). Proteins were then transferred to a protein blotting
membrane. T7 RNA polymerase was used to transcribe the full-length
TIN-ag cDNA into mRNA, which was in vitro translated into
protein using a rabbit reticulocyte lysate system (Stratagene). Lane 1, purified 58-kDa rabbit TBM TIN-ag positive control; lane 2, T7 RNA polymerase negative control; lane 3, T7 RNA polymerase TIN-ag reaction; lane 4, IgG negative
control. Detection was carried out as follows. Membrane was blocked in
PBS with 3% skim milk at 4 °C overnight. Primary antibody A8 was
incubated for 1 h at room temperature. Horseradish
peroxidase-conjugated goat anti-mouse secondary antibody, used for
detection by enhanced chemiluminescence (Amersham Corp.), incubated at
room temperature for 30 min. The membrane was washed before the
detection procedure was initiated, and the blot was exposed to film
(Amersham ECL film).
-terminal region of the
molecule contains a highly conserved epidermal growth factor-like
repeat common to several classes of extracellular matrix adhesive
glycoproteins, whereas the COOH-terminal region shares extensive
homology with the cathepsin family of cysteine proteinases. The spacing
of the two putative domains may indicate that TIN-ag arose by the
fusion of genes of extracellular adhesion glycoproteins and of cysteine
proteinases, although the possibility of convergent evolution cannot be
excluded.
-terminal sequence (24) is not found in
the predicted sequence of TIN-ag, suggesting that these two
nephritogenic antigens represent different macromolecules. The
possibility cannot be excluded, however, that alternative processive
forms of the same nephritogenic antigen are present in the TBM.
/EMBL Data Bank with accession number(s)
U24270.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.