(Received for publication, October 2, 1996, and in revised form, March 31, 1997)
From the Tokyo Institute for Immunopharmacology,
Inc., and Chugai Pharmaceutical Co. Ltd., Tokyo 171, Japan, the
¶ Gastroenterology Division, University of Colorado, Health
Sciences Center, Denver, Colorado 80220, the § Department of
Internal Medicine, School of Medicine, Keio University, Tokyo 160, Japan, and the
The Tokyo Metropolitan Institute of Medical
Science, Tokyo 113, Japan
Cloning a cDNA for human IgGFc binding
protein (FcBP) from human colonic epithelial cells reveals an
mRNA and coding region of 17 and 16.2 kilobases, respectively. The
predicted amino acid sequence contains 12 occurrences of a 400-amino
acid cysteine-rich unit resembling that found in mucin. A motif
(CGLCGN) in Fc
BP is conserved in MUC2 and prepro-von Willebrand
factor. The N-terminal 450-amino acid sequences are necessary and
sufficient to confer IgG Fc binding activity. Fc
BP mRNA is
expressed only in placenta and colonic epithelial cells. These results
suggest that Fc
BP may play an important role in immune protection
and inflammation in the intestines of primates.
Each antibody isotype has specific biological activities that are
dependent on the Fc receptor (FcR)1 it
binds (1, 2). For example, immunoglobulin G (IgG) complexes evoke
numerous functions. They are phagocytosed by macrophages, activate the
lytic potential of cytotoxic lymphocytes and monocytes, and regulate B
lymphocyte activation via different receptors for IgGFc (FcR)
expressed on different cells (3, 4). Soluble forms of FcR, called
immunoglobulin-binding factors, may also be involved in regulatory
functions (5).
Other types of immunoglobulin-binding proteins are involved in transport of immunoglobulins. For example, poly-immunoglobulin (Ig) receptors have been implicated in the transcytosis and secretion of polymeric IgA and IgM in mucosal epithelial cells (6, 7). Further, neonatal FcR (FcRn) expressed on the apical cell surface binds IgG in milk for endocytosis and subsequent transcytosis to the basolateral cell surface (8-12).
Recently, binding sites specific to the Fc portion of IgG molecules
have been reported in human intestinal goblet cells (13, 14).
Monoclonal antibodies K9 and K17, which block the binding to the Fc
portion of IgG, react with antigens >200 kDa in immunoblot analysis
using non-reduced colonocyte lysates. Under reducing conditions, K17
monoclonal antibody also reacts with proteins around 70-80 kDa. The
binding site is immunologically distinct from the known FcR on
leukocytes, is present in mucous granules, and appears to be secreted
with mucus into the intestinal lumen (14). In active ulcerative
colitis, a marked increase of IgGFc-binding sites in the endoplasmic
reticulum and a decrease in mucous granules are observed, apparently
reflecting increased synthesis and
secretion.2
Using monoclonal antibodies K9 and K17, we have cloned a full-length
cDNA of about 17 kb, which encodes an IgG Fc binding protein
(FcBP). Expression of an Fc
BP cDNA fragment (about 8 kb)
confers IgG Fc-binding activity in both COS and CHO cells. Our studies
reveal that Fc
BP shares some structural features with mucin
proteins. So far, the complete primary sequences of mucins MUC1
(15-17), MUC2 (18), and MUC7 (19) have been reported, but only partial
sequences are available for most other mucins because of tandem repeats
of sequences. Notably, the mucin-like protein we characterize here has
the biological activity of IgG binding, suggesting that it has an
important role in mucosal immune system and inflammation.
Human colonic or ileal epithelial cells were isolated as
described previously (13). The epithelial cells were pelleted and resuspended in PBS() and centrifuged at 4 °C for 10 min. Total RNA
was prepared with the method of Chomczynski and Sacci (20). Poly(A)+ RNA for the cDNA library construction was
prepared by affinity purification with oligo(dT)-latex beads
(oligotex-dT30, Takara Shuzo Co.). cDNAs were synthesized from the
poly(A)+ RNA using random primers and
oligo(dT)12-18 primer by Moloney murine leukemia virus
reverse transcriptase, as described by the manufacturer (cDNA kit,
Amersham Life Science, Inc.). Then,
EcoRI-NotI-BamHI linkers (Takara Shuzo
Co.) were ligated with the cDNAs. A random primed cDNA library
for screening with monoclonal antibodies was fractionated in size
greater than 500 bp, with an average of about 1.5 kb, and constructed
into the EcoRI site of
gt11 vector (Stratagene). The
cDNA library was screened by standard procedures (21) with monoclonal antibody K9 or K17 directed against colonic Fc
BP. Restriction endonuclease fragments (q and a/b as shown in Fig. 1B) from the isolated cDNA clones were used as probes to
isolate longer cDNA clones from the
gt10 library constructed as
follows. Poly(A)+ RNA prepared from human colonic
epithelial cells was used for synthesis of random primed or
oligo(dT)-primed cDNA as described above. Then the cDNA was
size-selected on agarose gels (>3 kb) and used to construct
gt10
libraries for the screening with DNA probes. Subsequent screening was
carried out with the unamplified library. 2 × 104
phage clones were plated on Luria-Bertani agar dish (10 × 13 cm),
and the plaques were transferred onto Biodyne A nylon filter (Nippon
Genetics Co.). The filters were hybridized with probe q and probe a/b
in a solution of 50% formamide, 5 × SSCP, 1 × Denhardt's
solution, 0.5% SDS, 100 µg/ml denatured salmon sperm DNA at 42 °C
overnight. Filters were washed three times with 0.2 × SSC
containing 0.2% SDS at 65 °C for 40 min and exposed to x-ray film
at
80 °C overnight. For DNA sequencing, a nest of insert unidirectionally deleted from each end was generated by exonuclease III
and mung bean nuclease digestions (Kilo sequence deletion kit, Takara
Shuzo Co.) as described in the manufacturer protocol. DNA sequencing of
double-stranded plasmid DNAs and single-stranded DNAs rescued with
VCSM13 helper phage was performed with standard dye primer-labeled
cycle-sequencing techniques using an Applied Biosytems 373A DNA
sequencer, and, in some experiments, internal sequencing primers were
synthesized with a DNA synthesizer Model 394 (Applied Biosystems) for
sequencing. Gene Works 2.3 (IntelliGenetics, Inc.) Macintosh software
was used for the DNA sequencing analysis.
Northern Blot Analysis
20 µg of total RNA prepared from colonic epithelial cells or from the human HT-29-18-N2 (designated as N2) cells was subjected to denaturing formaldehyde-agarose gel electrophoresis, transferred, and fixed onto a nylon membrane. The blots were prehybridized in the same solution as described in the screening method. Hybridization was performed with probe a, q, or y in the same solution at 42 °C overnight. The blots were washed 3 times with 0.2 × SSC and 0.2% SDS at 65 °C and exposed to x-ray film.
Zoo Blot AnalysisHigh-molecular-weight DNA was isolated from human colon epithelial cells by the methods of Nelson and Krawetz (22). Animal genomic DNAs were purchased from CLONTECH. Each DNA (5 µg) was digested with restriction enzyme EcoRI, electrophoresed in 0.7% agarose gel, and transferred to nylon membrane under alkali conditions. The filter was hybridized with a 32P-labeled 700-bp fragment of probe a.
Screening of 5EMBL3 SP6/T7 human leukocyte genomic library
(CLONTECH) was screened by plaque hybridization
using a 32P-dCTP random primer-labeled 908-bp
BamHI fragment of clone NZ4 and a 32P-5
end-labeled synthetic 33-mer nucleotide RPS1
(GCTCCAGCCCAGAGTATCCACCAGCTCCATAGG, which is complementary to
nucleotide sequences at the position of +17 to +49 of the NZ4 cDNA)
as a hybridization probe. Hybridization was performed at 42 °C in a
hybridization solution containing 5 × SSPE, 50 (for pNZ4 probe)
or 20% (for RPS1 probe) formamide, 2.5 × Denhardt's solution,
0.1% SDS, and 100 µg/ml heat-denatured salmon testis DNA. Filters
were washed five times for 15 min in 0.2 × SSC with 0.2% SDS at
65 °C (for NZ4 probe) or 0.5 × SSC with 0.1% SDS at 55 °C
(for RPS1 probe). Phages yielding positive signals on duplicate filters
were purified, and the fragments digested with restriction enzymes,
including XhoI and SacI, were subcloned into
pBluescriptSK(+).
Antisence 50-mer (5-GCTGATAGTTCTGCAGGAAGGCTGTGAGGAATTCC
TCTCTGGCCAGTGTTC) and 33-mer (5
-GCTCCAGCCCAGAGTATCCACCAGCTCCATAGG-3
) oligonucleotides, respectively complementary to nucleotides +95 to +144 and +17 to +49, were synthesized and purified by using an OPC
cartridge (Applied Biosystems) followed by polyacrylamide gel
electrophoresis. The 50-mer oligonucleotide primer was end-labeled with
[
-32P]ATP using T4 polynucleotide kinase (Takara Shuzo
Co.) and was used for the extension of cDNA from 2.5 mg of
poly(A)+ RNA, which was isolated from either N2 cells or
human colon epithelium cells using Moloney murine leukemia
virus-reverse transcriptase (Toyobo Co.). The extension products were
analyzed on a denaturing 6% polyacrylamide gel. A universal primer was
used to prime a sequencing ladder from single strand M13mp18 template
as size markers. For preparation of S1 probe, the end-labeled 33-mer
oligonucleotide was annealed to single-stranded DNA that was derived
from XhoI/EcoRI fragment of GHFc and contained
the 5
-flanking region of Fc
BP gene, extended with BcaBest
polymerase (Takara Shuzo Co.) at 60 °C for 10 min, and digested with
BamHI. The resultant double-stranded S1 probe was purified
by polyacrylamide gel electrophoresis. The 50,000 cpm of the S1 probe
was hybridized at 45 °C with 50 µg of total RNA isolated from
colon epithelium cells, and the mixture was subjected to S1 nuclease
digestion.
Polymorphic
SmaI restriction site of the FcBP gene was analyzed in
four cases of normal part of the colon epithelium from patients
harboring colon cancer and in 6 cases of normal blood lymphocytes. The
PCR reaction mixture contained 0.5-1 µg of DNA, 200 mM
each dNTP, 50 mM KCl, 20 mM Tris, pH 8.4, 2 mg/ml bovine serum albumin, 1.5 mM MgCl2, 0.5 units Taq polymerase (Perkin-Elmer Cetus), and 20 pmol of
each primer set in a total volume of 50 ml. The following
oligonucleotide primers for PCR were synthesized: BC1 (forward
5
-ACCACTCCTTCGATGGCC-), GS4R (reverse 5
-TGGTGCCGAGGGCAGCCACG-3
), GS1
(forward 5
-ACCTGTAACTATGTGCTGGC-3
), and GS3R (reverse
5
-ACAGCAGGGTTGCCCCGG-3
). Reverse-transcription PCR (RT-PCR) was also
carried out. Total RNA (0.6 µg) from N2 cells was used as starting
material. The first strand synthesis was performed at 60 °C for 30 min using avian myeloblastosis virus reverse transcriptase with primer
GS3R or GS4R followed by inactivation of the enzyme at 95 °C for 5 min, and then each forward primer was added to the mixture. The thermal
cycle profile for both PCR and RT-PCR was as follows: 95 °C for 1 min, 60 °C for 1.5 min, 72 °C for 2.5 min for 30 cycles. Each 7 ml of the amplification product was digested with 50 units of
SmaI in a total volume of 10 ml at 30 °C for at least
12 h. The digested-amplification products were electrophoretically
separated on 2% agarose gels and visualized by ethidium bromide
staining under UV-light. Quantitative determination of products was
carried out using scan analysis software with photographs of the
gels.
pcDL-SR296 vector (23)
was kindly provided by Dr. Takebe of the National Institute of Health
(Japan). pMSXND vector (24) contains metallothionein promoter for
foreign cDNA expression and dhfr gene for selection of
clones and gene amplifying.
Plasmids to
express about half of full-length cDNA were constructed as follows.
Five cDNA fragments (see Fig. 1) were used for construction of
expression cDNA. The fragment of nucleotide 1 (5 end) to
nucleotide 313 (BglII) was derived from clone NZ4, nucleotide 314 (BglII) to nucleotide 1244 (BstXI)
was from C72, nucleotide 1245 (BstXI) to nucleotide 1664 (HincII) was from Y11, nucleotide 1665 (HincII)
to nucleotide 4442 (BamHI) was from X1, and nucleotide 4443 (BamHI) to nucleotide 7784 (3
end of the clone) was from
V11. The fragments were ligated to each other, and the ligated 7.9-kb
cDNA fragment was inserted at the cloning site of pcDL-SR
296,
which was under control of the SV40 promoter, to yield pSR-NV11.
The deletion constructs were prepared as follows. (i)
The 5 end-BglII fragment of NZ4 clone was ligated to the
BglII site of C72 clone to produce NZ. (ii) NZC was ligated
to the ~420-bp fragment of Y11 clone at the BstXI site to
yield NZCY. (iii) The XhoI (cloning site on a
vector)-BstXI fragment from NZC was ligated to the
BstXI-HincII fragment from Y11, and the resultant
1.7-kb fragment was inserted in the HincII-digested X1 clone
to produce NX. (iv) NV11 was recovered from the plasmid by digestion
with NotI and inserted in pUC119, of which the
HincII site changed to NotI site to generate
pUC-NV11. pUC-NV11 was digested with HincII,
BssHII, Tth111I, or SplI and
SpeI, respectively, and each digest containing vector
sequences was self-ligated to produce
Hinc,
BssH,
Tth, or
Spl,
respectively. (v)
Tth, NX, and
Hinc were digested with BssHII, and then each
digest containing vector sequences was self-ligated, respectively, to
generate
BssH/Tth, NX,
BssH, and
Hinc/BssH. (vi) NotI-digested
insert of V11 was blunted by Klenow fragment and was inserted into the
blunted SpeI site of NZC to produce NZCV11.
For expression, the original initiation site of each construct was
alive except for X1. To express clone X1, an oligonucleotide adapter
containing the initiation site sequences originated from FcBP
cDNA (or NV11), intervened between HindIII- and
EcoRI-site sequences, was designed as follows
(5
-AAGCTTCTGCAGCCATGGGGGATCC-3
). The oligonucleotide was inserted
into HindIII-EcoRI site of pBLS-STOP to make
transcription start followed by cDNA sequences. The insertion sequences of clone X1 digested with EcoRI were ligated into
EcoRI site of pBLS-STOP to produce X1. Stop codon sequences
were also designed as follows. The two adapter sequences were
synthesized (upper strand 5
-CTAGTTAGTTAGTTAGGGTACCGC-3
and lower
strand 5
-GGCCGCGGTACCCTAACTAACTAA-3
). This double strand provided
stop codon in all frames. It was inserted between SpeI and
NotI site of pBluescript II SK(+), followed by modification
of cloning site of XhoI to XbaI site to yield
pBLS-STOP vector. All constructs encoding Fc
BP derivatives were
cloned into pBLS-STOP, and the insertion sequence containing stop
codons was subcloned into pcDL-SR
296 for protein expression. The
expression vectors containing these deletion constructs were
transfected transiently in COS7 cells, and produced proteins were
stained with Fc fragment of human IgG or monoclonal antibodies K9 and
K17.
For transient expression, COS7 cells were
grown in RPMI 1640 medium containing 10% fetal bovine serum (FBS),
10,000 IU/liter penicillin, and 100 mg/l streptomycin. COS7 cells were
plated at 1 × 105 cells/35-mm tissue culture dish
a day before transfection. Then the cells were transfected with pSRNV11
by lipofection procedure using 10 µg of DNA and 5 µl of Transfectam
(Promega) per 500 µl of RPMI 1640 medium. After 6 h, medium was
changed and cultured for a further 48 h. For permanent expression,
CHO cells were transfected with a pMSXND vector bearing FcBP
cDNA fragment, NV11, or the deleted cDNA by lipofection. Cells
were passaged the day before transfections, and the subconfluent 2 × 105 cells were treated with CsCl-purified plasmids in
the same way as COS cells. The cell medium was replaced with F-12
medium supplemented with nucleotides and 10% FBS and incubated at
37 °C for 2 days. Then the cells were selected in
-minimum
Eagle's medium without nucleotides containing 1 mg/ml G418, 10% FBS,
and antibiotics. After culturing under these conditions for 14 days,
the clonal cell lines were obtained by limiting dilution and were
examined for Fc
BP expression by immunostaining. Then the clonal cell
lines were propagated with increasing concentrations of methotrexate, starting with 0.02-6.4 µM. The gene-amplified cells were
confirmed by a higher amount of expression of Fc
BP, and clonal cells
were obtained by limiting dilution.
Random primed cDNA libraries (>600 bp) were
constructed from human colonic epithelial cells using gt11 as a
vector. The
gt11 human epithelial cell library was first screened
with two independent monoclonal antibodies against Fc
BP, K9, and K17
that block the binding of IgG Fc fragment (14). The screening of 1 × 106 recombinants with monoclonal antibody K9 led to the
isolation of a cDNA clone 618 bp long (Fig.
1B) in the region defined by nucleotide
position 13788-14405 (probe q). Furthermore, the screening of 6 × 105 recombinants with monoclonal antibody K17 led to the
isolation of 7 clones, one of which was about 1300 bp long and could be digested with BamHI into 2 fragments (a and b). These
adjacent fragment probes (a/b) were corresponding to nucleotide
positions 7368-8045/8046-8697 or 10970-11649/11650-12300,
respectively. The nucleotide sequences of probes q and a/b revealed
that they are independent cDNA clones. However, both probes
hydridize to a single band of >16 kb on a Northern blot (described
below). These results suggest that the two clones are derived from the same mRNA for Fc
BP. Three probes, q, a, and b, were used to
determine the full-length cDNA sequence. Out of more than 70 clones
isolated, 10 clones (T5, A43, A8, A31, A40, A53, V11, X1, Y1, and C72)
were shown to cover most of full-length cDNA for Fc
BP except for
5
-terminal sequence (Fig. 1C).
Sequence analysis of these clones showed that the FcBP cDNA is
composed of three homologous units that are tandemly repeated (see Fig.
1B). Probe q hybridizes with 3 regions, as does probe a/b.
Each unit shares more than 95% homology with one another. Although a
cDNA clone representing the 3
-terminal region, T5, was isolated
from oligo(dT)-primed cDNA library, no 5
-terminal clones
(i.e. extending from C72) were isolated from human colonic libraries. Thus, we constructed a cDNA library of human N2 cell line, which can differentiate into goblet cells and express Fc
BP (25, 26). Using a 5
-terminal probe derived from the C72 clone, we
obtained a clone, NZ4, containing the 5
-terminal ATG. Sequences flanking the first ATG (GCC(A/G)CCATGG) in NZ4 clone are
consistent with those described by Kozak (27) for an initiation codon. The complete nucleotide sequence data has been submitted to the DDBJ,
EBI, and GenBankTM Data Banks; a map of the major
restriction enzyme sites is shown in Fig. 1A.
A full-length sequence of the
predicted amino acids is shown in Fig. 2A. A
dot matrix plot of the entire predicted amino acid sequence against
itself revealed 12 repeated domains flanked by unique N-terminal (450 amino acids) and C-terminal (160 amino acids) domains (data not shown).
These 12 repeated domains (r1-r12 in Fig. 2B)
were classified into 5 types that shared 30-40% homology. The R1 unit
(r3-r5) corresponds to the first 5-terminal A-B-C-Q region in Fig. 1.
The repeated domains r1-r12 are each composed of about 400 amino
acids.
As some mucin-related proteins have been reported, the predicted amino
acid sequence of human FcBP was compared with those amino acid
sequences, and a broader search for similarities to other protein
sequences was carried out using Gene Works (IntelliGenetics, Inc.)
loading GenBankTM Release 8.7. These analyses demonstrate
that the Fc
BP sequence has significant similarity to portions of
MUC2 and prepro-von Willebrand factor (vWF) but that it does not have
homology to either Fc receptors or IgG-like domain. A close examination
reveals that each repeated domain of Fc
BP was homologous with four
MUC2 D domains and to four prepro-vWF D2 domains (about 30% homology). Interestingly, as shown in Fig. 3, Fc
BP, vWF, and
MUC2 all have the conserved amino acid motif CGLCGN. These sequences
are also characteristic of thioredoxin (28) and protein disulfide
isomerase (29). Another feature of this protein is that it is high in cysteine content. As shown in Fig. 2B, 8.1% of the amino
acid residues are cysteines (cf. 5.7% for human serum
albumin (hSA)). A total content of the predicted serine/threonine
residues for O-linked glycosylation is 12.3% (Fig.
2B, cf. 9.3% for hSA). Further, the content of
hydrophobic and neutral amino acids is 78.6% (cf. 63.8%
for hSA).
Size Determination of Native mRNA for Human Fc
Northern blot analysis was carried out to detect the
expressed mRNA for FcBP from colonic epithelial cells. Probes q,
a, or y (an 800-bp probe derived from the 5
-terminal region of clone Y1) were used. A single band of larger than 15 kb with a smeared front
was detected with each probe, as shown in Fig.
4A. Probe y gave the same Northern blot
pattern as with probes q and a, indicating that these three probes
hybridize to the same mRNA. The same result was obtained using a
part of clone NZ4 as a probe (data not shown).
As described above, however, mRNA size is critical for determining
the number of 4.5-kb units (A-B-C-Q). To determine more precisely the
size of the FcBP mRNA, we compared it with mRNAs for
ryanodine receptor (15.2 kb) and for dystrophin (14 kb), which are
among the longest mRNAs reported. We prepared the respective probe
for them by PCR method, using poly(A)+ RNA from human
cytoskeletal muscle. Since Fc
BP has been shown to be present in
human N2 cells by immunohistochemical staining with monoclonal
antibodies K9 and K17 (26), we carefully prepared the mRNA from the
N2 cells. As shown in Fig. 4B, Northern analysis using probe
a showed that the size of our mRNA was estimated to be 17 kb by
relative mobility of two standard mRNAs. These results confirmed
the fact that a 4.5-kb-long unit was repeated 3 times, in addition to
5
-terminal and 3
-terminal unique sequences.
To confirm that the 5-terminal
cDNA sequence of human Fc
BP is identical to genomic DNA,
approximately 2 × 106 recombinant phages from a human
leukocyte genomic library were screened using the 5
-terminal fragment
of clone NZ4 as a probe. One independent clone, GHFc1, and two
overlapping clones, GHFc2 and GHFc3, were identified. A 1908-bp
SacI/EcoRI fragment at the 5
-flanking region
from GHFc1 as well as the exon 2/intron boundary regions from GHFc2 and
GHFc3 were subcloned into pBluescript and sequenced using exonuclease
III-generated deletion templates.
Fig. 5 shows the sequence of the first and second exons
of genomic DNA, which completely coincides with the corresponding region of cDNA. The exon/intron splice sites were confirmed to be
GT-AG border element consensus sequences. The first exon contains the
putative ATG initiation codon suitable for Kozak's rule (27), as
described above. Examination of the 5 untranslated region reveals a
TGA in-frame stop codon at
78 position of the gene. However, no TATA,
CAAT, or other promoter/enhancer motifs were detected within 2 kb
further upstream (data not shown).
To identify the transcription start sites, primer extension and S1
mapping were also performed. Primer extension analysis using an
oligonucleotide complementary to the sequence double-underlined in Fig.
5 reveals multiple start sites (see Fig. 6A).
Three major bands are located at +27, +28, and +30 nucleotides
downstream. The upper broad band is located at 5 to +25 in a boundary
region of 5
terminus for cDNA clone NZ4. Similar results are
observed using human colon epithelial cells, except for additional
faint bands. Clusters of multiple start sites are common for genes
lacking a TATA box (30).
As shown in Fig. 6B, the end-labeled 205-bp fragment hybridizes with total RNA from human colon epithelial cells protected from S1 nuclease digestion, and several hybrids are detected. These products also reveal multiple start sites, and the longest digestion showed the predominant start site to be an adenosine residue that is only 9-bp upstream from the deduced ATG translation initiation site. Thus, the same transcription start sites are suggested by the broad extension band seen in primer extension analysis.
Distribution of SmaI RFLPTo detect sequence variants and to
study whether those variants are allele-specific or repeated
region-specific in each allele, RFLP analysis with SmaI was
investigated for 10 individuals and cultured N2 cells. We designed two
distinct primer sets, as described under "Materials and Methods."
The PCR-amplified products are 186 bp (primers BC1 and GS4R) as shown
in Fig. 7 and 107 bp (primers GS1 and GS3R) (data not
shown), and the complete digestion of these products with
SmaI was followed by electrophoresis. The digestion with
SmaI results in fragmentation into 113 and 73 from 186 bp
and into 72 and 35 from 107 bp, respectively. In the case of sample 4, only 113- and 73-bp SmaI-digested bands were observed, but 9 other cases showed the additional band (186 or 107 bp) non-digested with SmaI. Using primers GS1 and GS3R, consistent results
were obtained by detecting a set of 72- and 35-bp fragments after
SmaI digestion.
Reflecting the sensitivity to SmaI digestion, we determined
two alternative sequences. One was present in clones of A53, A8, and
others (ACT-GGC-TGC-CCC-GGG-GGT), which are sensitive to
SmaI, and another sequence (ACT-GGC-TGC-CTG-GGG-GGT) present
in clones of V11 and others, which are resistant to SmaI.
This difference in sequence results in changing one amino acid residue
of proline to leucine. As FcBP gene has three large repeats, six
large repeats are involved in a diploid genome. Although theoretical
ratios of SmaI-digested bands to undigested bands are 6:0,
5:1, 4:2, 3:3, 2:4, 1:5, or 0:6, no case in which all 6 repeated
regions are SmaI-resistant has yet been observed. RT-PCR of
mRNA from N2 cells reveals the message to be all
SmaI-sensitive (Fig. 7, sample RT).
Human
FcBP mRNA expression in various human tissues was examined.
17-kb mRNA is expressed in the placenta and colonic epithelial cells, but no expression was detected in heart, brain, lung, liver, skeletal muscle, or kidney (Fig. 8). Since IgGFc binding
activity has not yet been detected in non-human mammals such as mice
and rabbits (13), we investigated the species specificity of Fc
BP gene. The gene for Fc
BP was detected in human and monkey but not in
mouse, rabbit, rat, dog, bovine, or porcine by zoo blot analysis (data
not shown) even when hybridization was carried out under several
diffferent conditions (data not shown).
Expression of Active Recombinant Molecules
The large size of
the intact cDNA for human FcBP precluded expression in a
standard expression vector. Therefore, COS cells were transfected with
a cDNA fragment containing the H domain and only one A-B-C repeat
unit. About 30% of transfected cells produced proteins that reacted
with monoclonal antibodies K9 or K17 against Fc
BP, compared with
nontransfected cells (Fig. 9, panels a,
b, and c). We then examined whether transfected
COS cells had IgG binding activity using IgG conjugated with
horseradish peroxidase (HRP). The fraction of transfected COS cells
bound to HRP-IgG was almost the same as that stained with monoclonal antibodies (Fig. 9, panel d). Furthermore, a similar
fraction of transfected COS cells bound IgG Fc fragment, whereas IgG
F(ab
)2 failed to bind to the cells. We also examined the
binding of IgM, IgA, and secreted IgA to the transfected cells. None
bound to the cells (data not shown). These results show that the NV11
cDNA fragment for human Fc
BP is sufficient to express a
biologically active protein fragment (designated as Fc
BPf) that can
bind IgG Fc but not IgG F(ab
)2.
Inhibition of IgG Binding by Heat-aggregated IgG Using Fc
We isolated a CHO stable
transfectant clone expressing FcBPf. More than 90% of cells
produced the protein product. Therefore, we could determine the binding
activity quantitatively using HRP-conjugated human IgG. Fig.
10 shows the competitive inhibition of HRP-labeled IgG
binding in the presence of the various concentration of human monomeric
IgG or heat-aggregated IgG. Monomeric IgG can specifically inhibit the
binding of HRP-conjugated IgG, and heat-aggregated IgG shows 10 times
stronger inhibition than monomeric IgG. These results suggest that
polymeric IgG, like the heat-aggregated form, has a higher affinity to
Fc
BPf than monomeric IgG.
Functional Analysis of Fc
Subsequently, we
prepared nested deletions of NV11 cDNA to identify regions of
cDNA essential for biological activity (Fig. 11).
Monoclonal antibody reactivity and IgG binding activity of the protein
fragment produced in the transfectants are also summarized in Fig. 11.
Notably, clones deleted within the H region (Hinc,
Hinc/BssH, or X1) can express proteins with
monoclonal antibody reactivity but no IgG binding activity. These
results suggest that the subregion from r1 through r5 (Fig. 11) is
responsible for IgG binding and that the H region is essential for
expression of functional molecules. Although about half the r5 domain
is deleted in clone
Spl, its reactivity with monoclonal antibody K9
is retained. In clones
Tth and
BssH/Tth, K17 reactivity is attributable to a
part of the r6 domain, which is highly homologous to the r3 domain.
Prediction of IgG Binding Sites
Domains r1-r5 show 30-40%
amino acid sequence homology with one another. We tried to determine
whether each domain is responsible for IgG binding. COS cells
transfected with the deleted cDNAs were treated with HRP-IgG for
IgG binding assay and also incubated with the HRP-IgG in the presence
of excess unlabeled competitors, monoclonal antibodies K9 and/or K17.
When some r domains were deleted (e.g. clones NX and NZCY in
Fig. 11), the strength of IgG binding was proportional to the number of
intact r domains; a clone completely lacking r domains (such as NZC)
showed no IgG binding activity. Since monoclonal antibodies K9 and K17
interact with the r3 and r5 domains, respectively, K9 and K17
competitively inhibited the IgG binding activity of clones expressing
FcBP containing r3 and/or r5 domains (Fig. 11). These findings
suggest that at least r1, r3, and r5 domains are involved in the IgG
binding capacity of Fc
BPf.
Our cDNA sequence
reveals that full-length FcBP comprises 5405 amino acid residues
(Fig. 2). Based on its structural features, this protein can be divided
into three major domains. The largest central domain is composed of 12 tandem repeats of about 400 amino acids each (Fig. 2B). Each
repeat contains about 8% cysteine residues. Repeats are homologous
(about 30-40%) to each other. The lability of IgG binding activity in
periodic acid and hydrogen peroxide treatments (13) and broad bands
observed on SDS-PAGE gels suggested that Fc
BP is a highly
glycosylated protein. The presence in the predicted amino acid
sequences of many N- and O-linked glycosylation sites (15.5%) is consistent with this. The structure of Fc
BP is
related to the mucin-like protein MUC2. Taking together its intracellular transport and localization in goblet cells (14), this
fact indicates that Fc
BP may be a component of mucus.
Gel-forming mucin is thought to be a giant molecule formed by several
to tens of molecules bound by inter-cystine bonds (31). The tandem
repeat domain, for example, composed of repeats of 17-amino acid units
for MUC3 (32), 16 for MUC4 (33), and 169 for MUC6 (34) is
characteristic of conventional mucin. However, no such domain
containing short repeat units was detected in the molecule of FcBP.
Nevertheless, Fc
BP should be classified as one of the mucins because
of the fact that (i) it has a high molecular weight (>200 kDa) with
S-S linkages (13), (ii) it is secreted with mucus from goblet cells
into the intestinal tract (14), (iii) it may be glycosylated (13), and
(iv) it contains several cysteine-rich domains (Figs. 2 and 3). Thus,
Fc
BP appears to be a mucin-like protein and to be involved in the
maintenance of the mucosal structure as a gel-like component of the
mucosa.
We analyzed cDNA libraries prepared
from human tissue and the N2 cell line, and detected several different
types of FcBP cDNA, as assayed by SmaI sensitivity.
Since a difference was specifically seen in the SmaI site of
highly homologous 3.6-kb DNA repeats, we focused on the analysis of
polymorphisms of this site. This analysis revealed several particular
digestion patterns dependent on the number of SmaI sites,
including polymorphisms of each allele in the Fc
BP gene (Fig. 7).
This finding also suggested the expression of Fc
BP gene in different
alleles in individuals. No major variations in the Fc
BP gene, such
as variations in the number of homologous repeats or in splicing, have
been detected to date (Fig. 4). While the amino acid residue of the
SmaI site was changed from proline to leucine or reverse, it
is unknown whether polymorphism of the Fc
BP open reading frame
caused by point mutation alters the Fc binding activity of Fc
BP
related to colorectal diseases, as is the case for familial intestinal
polyposis and polymorphisms of its causative gene (adenomatous
polyposis coli gene) (35). The physiological function of gene
polymorphisms in Fc
BP remains to be elucidated.
The protein FcBPf produced by the
expression of an approximately 8-kb fragment including the 5
terminus
of Fc
BP cDNA (NV11 clone) contained an H domain and 6 r
domains (Fig. 11). To identify the domain possessing Fc binding
activity, we assessed activity using a series of deletions in cDNA.
When some of the r domains were deleted, IgG binding tended to become
weaker in proportion to the length of the remaining r domains. The
deletion experiments also showed that r5 domain is critical for IgG
binding. Competition experiments using inhibitory monoclonal antibodies
suggest r3 and r5 domains are involved in the binding of Fc although
the r1 domain of clone NZCY still shows significant binding activity. Staining with monoclonal antibodies revealed that K9 recognizes the r5
domain, as well as r11, and that K17 recognizes the r3 domain, as well
as r6 (Fig. 11). Our results further suggest that both the r3 and r5
domains possess independent Fc binding sites as well as r1 domain.
Therefore, we speculate that at least three IgG molecules can bind to a
single Fc
BPf protein molecule. Furthermore, the inhibition of IgG
binding by heat-aggregated form implies that the polymeric form such as
immune complexes may be a better ligand for Fc
BP.
Considering the lower cysteine content of H domain and its unique amino
acid sequences, it is likely that it may differ in nature from r
domains. Deletion of the H domain results in loss of Fc binding
activity (Fig. 11). This suggests that the H domain plays an important
role in maturation of biologically active protein products. We
speculate that H domains are involved either in the processing of the
FcBP polypeptide into an active form with Fc binding activity or in
the intracellular localization for suitable protein processing, such as
Golgi apparatus or mucus granules. These speculations seem to be
reasonable because the following facts were observed. Although the
predicted molecular weight of the whole Fc
BP (about 5,000 amino
acids) is more than 500 kDa, Kobayashi et al. (14) detected
a more than 200 kDa band by non-reduced electrophoresis and 70-80 kDa
bands under reduced conditions. Thus, the Fc
BP may be processed by
protease after translation. So far, our Fc
BPf expressed in COS and
CHO cells is also recovered as broad bands of 50-80 kDa under reduced
conditions by Western blot analysis.3
Our previous study using
monoclonal antibodies showed the presence of FcBP in the mucosa of
the large and small intestines (13, 14). In the present study, Fc
BP
mRNA is detected not only in the colorectal epithelial cells but
also in the placenta (Fig. 8). These findings imply that Fc
BP may be
distributed in the systemic mucosa, as are the secreted mucins MUC2,
MUC3, and MUC4. Fc
BP was detected only in humans and monkeys by
Southern blotting. Therefore, Fc
BP may play a role in the mucosal
immune system of primates. IgA and IgM are transported within
epithelial cells via polymeric Ig receptors, and they eliminate toxic
antigens from the lamina propria. Analysis of the amino acid sequences of Fc
BP, however, reveals no membrane-penetrating domain or signal peptide sequences (Fig. 2). Immunohistochemical studies reveal that
Fc
BP is transported from Golgi apparatus to the mucous follicles (14) although it is unknown whether protein localization is dependent
on binding to IgG. It therefore seems unlikely that Fc
BP serves as a
transporter-like poly-Ig receptor or incorporates IgG-like FcRn.
However, similar to poly-Ig receptor, a portion of which serves as a
secretory component and contributes to multivalent IgA formation and
its stabilization, it is likely that Fc
BP enhances antigen trapping
through its promotion of multivalent IgG formation or protects IgG from
degradation by bacterial proteases. Further, secreted into the mucus,
native Fc
BP may prevent the invasion of antigens into the mucosa by
efficiently trapping antigen-IgG complexes through its binding to more
than nine of the aggregated IgG complexes. Further studies are being
conducted to reveal where Fc
BP is able to bind IgG molecules during
protein processing.
FcBPf expressed from an 8-kb cDNA are cleaved into several
polypeptides cross-linked by disulfide bonds.3 Intra- and
inter-disulfide bonds are also suggested by the conserved motif
(CGLCGN) in all the repeated domains (Fig. 3). Interestingly, such
vicinal cysteines are conserved in all members of thioredoxins, as well
as vWF and MUC2. Thioredoxins are involved in a wide variety of
biochemical systems, and the vicinal cysteines (CGPC for thioredoxins) are essential for redox functions in E. coli. In mammalian
cells, thioredoxin functions as an endogenous glucocorticoid
receptor-activation factor (36). Moreover, thioredoxin-like domains
have been found in several proteins of higher molecular weight, such as
protein disulfide isomerase (29) and phosphoinositide-specific
phospholipase C (37). Another interesting function of eukaryotic
thioredoxin is its stimulation of interleukin 2 receptor expression in
human T-cell leukemic virus (HTLV)-1 transformed T cells, an activity originally described as adult T-cell leukemia-derived factor (ADF) (38,
39). Recently, recombinant ADF prevented the cytotoxicity caused by
H2O2 (40). These results suggest that high
cysteine content, as well as the conserved sequences in human Fc
BP
molecules, serves as an anti-oxidant in mucus. This speculation led to
protective elimination of antigen-immune complex from the intestinal
tract.
Regarding the vicinal cysteines, another potentially interesting
parallel is the possibility that FcBP, like pro-vWF (41), may have
the ability to catalyze its own oligomerization. Pro-vWF autocatalysis
occurs in vitro at low pH and is dependent upon the
integrity of two sets of vicinal cysteine residues, both with the
sequence CGLC (42). These tetrapeptides apparently conduct thiol
oxidation through a mechanism involving the formation of a strained
14-member ring joined at the sulfur atoms of the two cysteine residues.
These sequences are conserved in each subdomain of Fc
BP. These
vicinal cysteines may be important in processing to an active form as
well as H domain, and in formation of structural networks by mucin
proteins.
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ and GenBankTM/EBI Data Bank with accession number(s) D84239[GenBank].
We thank Dr. Yutaka Takebe (National
Institute of Health of Japan) for providing pcDL-SR expression
vector and Dr. Shigeru Taketani for many helpful suggestions. We also
thank Tomoe Minami and Kazuko Kajiyama for technical assistance.