Cloning and Characterization of IL-1HY2, a Novel Interleukin-1
Family Member*
Haishan
Lin,
Alice S.
Ho,
Dana
Haley-Vicente,
Jun
Zhang,
Juanita
Bernal-Fussell,
Ann M.
Pace
,
Derek
Hansen,
Kathi
Schweighofer,
Nancy
K.
Mize, and
John E.
Ford§
From the Functional Genomics Department, Hyseq Inc.,
Sunnyvale, California 94086
Received for publication, November 6, 2000, and in revised form, March 13, 2001
 |
ABSTRACT |
The interleukin-1 (IL-1) family members play an
important role in the process of inflammation and host defense. We
describe here the identification and characterization of a novel member of the IL-1 family, IL-1HY2. The human IL-1HY2 protein shares significant amino acid sequence similarity (37%) with the IL-1 receptor antagonist and has a predicted three-dimensional
structure similar to that of the IL-1 receptor antagonist. The
IL-1HY2 gene is located in close proximity to other IL-1
family genes on human chromosome 2, and the genomic organization of the
IL-1HY2 gene is highly conserved with other IL-1 family
members. IL-1HY2 protein is secreted from mammalian cells, and the
purified recombinant IL-1HY2 protein binds soluble IL-1 receptor type
I. IL-1HY2 is expressed in human skin, spleen, and tonsil.
Immunohistochemical analysis showed that the IL-1HY2 protein is
expressed in the basal epithelia of skin and in proliferating B cells
of the tonsil. These data suggest that IL-1HY2 is a novel IL-1 family
member and that it may participate in a network of IL-1 family members to regulate adapted and innate immune responses.
 |
INTRODUCTION |
Interleukin (IL)-1
,1
IL-1
, IL-1 receptor antagonist (IL-1ra), and IL-18 belong to the
IL-1 family of cytokines. IL-1 cytokines have been implicated in a wide
range of pathological conditions such as rheumatoid arthritis,
inflammatory bowel disease, Alzheimer's disease, diabetes, and
susceptibility to infections (1, 2). The balanced expression of the
IL-1 family members has been postulated to be important in regulating
inflammation and host defense responses. IL-1
and IL-1
signal
through the IL-1 type I receptor, whereas IL-1ra functions as an
antagonist by competitively binding to the same receptor without
eliciting receptor signaling (1, 3). IL-18 signals through the IL-18
receptor, inducing the production of
-interferon from T cells (4).
Recently, five additional IL-1 family members, including IL-1HY1 (5,
6), FIL1
, FIL1
, FIL1
(7), and IL-1H1 (8), as well as some of
their isoforms (8, 9), have been reported.
Cytokines are often expressed at low levels owing to their potent
effects. To identify novel cytokine genes expressed at low levels, a
unique high throughput cDNA screening approach (10, 11) was
employed. This approach utilizes a set of oligonucleotide probes to
hybridize successively to arrays of human cDNAs from various
tissues. Hybridization patterns allow the cDNA clones to be
clustered into groups of similar or identical sequences. The number of
clones in a particular cluster represents the relative expression level
of a specific gene. Representative clones from clusters with low
numbers of members were selected for sequencing. Novel cytokine family
members were identified using various sequence analysis algorithms. The
IL-1HY1 gene was recently identified using this approach
(5).
In the current report, we describe the discovery of a novel IL-1 family
member referred to as IL-1HY2. The characteristics of
IL-1HY2 indicate that it shares a common ancestral precursor gene with other IL-1 family members. We demonstrate that IL-1HY2 is
secreted, binds the soluble IL-1 receptor, and is expressed in human
skin, spleen, and tonsil, suggesting that it may play a role as a
secreted ligand in host defense and other immune responses.
 |
EXPERIMENTAL PROCEDURES |
Isolation of the IL-1HY2 cDNA and Sequence Analysis--
A
cDNA library was constructed from human fetal skin mRNA
(Invitrogen) in the pSport1 vector (Life Technologies, Inc.). Inserts of the library were amplified by PCR using primers specific to pSport1
vector sequences that flank the inserts. These samples were then
analyzed using the screening by hybridization approach as
described previously (10, 11). Briefly, the PCR products were spotted
onto nylon membranes and hybridized with oligonucleotide probes to give
sequence signatures. The clones were clustered into groups of similar
or identical sequences, and single representative clones for rarely
expressed transcripts were selected for gel sequencing. The 5' sequence
of the amplified inserts was then deduced using the reverse M13
sequencing primer by ABD Big Dye terminator cycle sequencing
(PerkinElmer Life Sciences). The cDNA sequence data base was
analyzed using the BLAST and motif search algorithms. A novel cDNA
sequence encoding a polypeptide homologous to the IL-1ra protein was
identified. The sequence was confirmed in part by sequencing PCR
products generated from 5' rapid amplification of cDNA ends
analysis using a Marathon cDNA amplification kit (CLONTECH). This novel gene sequence was designated
IL-1HY2.
Chromosomal Localization and Genomic Organization of the IL1HY2
Gene--
PCR primers specific to IL-1HY2
(5'-CCGCACCAAGGTCCCCATTTTC-3' and 5'-GAGCCCACAAGGATAACCCAGG-3')
were used to screen the NIGMS human/rodent somatic cell hybrid
mapping panel 2 (12) and the Stanford G3 Human/Hamster Radiation Hybrid
panel (13) (Research Genetics, Huntsville, AL). A PCR product of 824 base pairs was amplified using the following conditions: 94 °C for 3 min; 30 cycles of 94 °C for 30 s, 58 °C for 30 s, and
72 °C for 1 min; and 72 °C for 10 min. Linkage analysis and
subsequent chromosomal localization were obtained using the Stanford
Human Genome Center RH server. To determine the genomic
structure of the IL-1HY2 gene, human BAC clones (Research
Genetics) were screened by PCR with primers specific to the IL-1HY2
cDNA. The BAC393I6 containing the IL-1HY2 gene was
partially digested by Sau3AI enzyme and size-selected restriction fragments were inserted into the BamHI site of
pUC18 (Amersham Pharmacia Biotech) to generate a library for
sequencing. Sequencing was performed using M13 forward and reverse
primers flanking the inserts. Direct BAC DNA sequencing was also
performed using the primers specific to IL-1HY2 cDNA to
confirm the exon/intron organization.
cDNA Expression Analysis--
Plasmid DNA (20 ng) of human
cDNA libraries derived from a range of tissues were used as
templates in a PCR reaction. Primers specific to IL-1HY2
(5'-AGGACCAGACACCACTGATTG-3' and 5'-TGGGGGCACAAGGCTAAAAC-3') and
-actin (5'-CCTAAGGCCAACCGTGAAAAG-3' and 5'-TCTTCATGGTGCTAGGAGCCA-3') were used. PCR was performed using the following conditions:
94 °C for 3 min; 30 cycles of 94 °C for 30 s, 58 °C for
30 s, and 72 °C for 1 min; and 72 °C for 10 min.
Transfection of Chinese Hamster Ovary (CHO) Cells and Protein
Analysis by Western Blot Analysis--
The coding region of the
IL-1HY2 cDNA was cloned into the pcDNA3.1/V5-His TOPO vector
(Invitrogen) using PCR primers
(5'-GAGCCGCCATGTGTTCCCTCCCCATGGCAAG-3' and
5'-GCTACCAGCTCTGTTCAAAGTAAAAC-3'). CHO cells obtained from the
American Type Culture Collection were cultured in F12K supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin sulfate at 37 °C in 5% CO2. CHO
cells were transfected with the IL-1HY2 expression vector or a control
empty vector using Fugene-6 according to the manufacturer's
instructions (Roche Molecular Biochemicals). After the transfection,
medium was replaced with serum-free F12K medium, and the cells were
incubated for an additional 24 h prior to harvesting. Conditioned
medium was centrifuged and then filtered through a 0.2-µm filter
(Pall Gelman Laboratory) to remove cell debris. The conditioned medium was subsequently concentrated by 10-fold using the Microcon YM-10 according to the manufacturer's instructions (Amicon). Cell lysates and conditioned media were analyzed by Western blot analysis. After
SDS-polyacrylamide gel (15%) electrophoresis, protein samples were
electroblotted onto an Immobilon-P membrane (Millipore) and probed with
the anti-IL-1HY2 antibodies. Immunocomplexes were visualized by
SuperSignal West Pico chemiluminescence detection reagents with goat
anti-rabbit IgG conjugated with horseradish peroxidase as a secondary
antibody (Pierce).
Purification of Recombinant IL-1HY2 Protein and Measurement of
Receptor-Ligand Binding Affinities--
The coding region of IL-1HY2
cDNA was cloned into pCR II/TOPO vector (Invitrogen) using PCR
primers (5'-GTCATATGTGTTCCCTCCCCATGGCAAG-3' and
5'-GAAGCTTGATCACTACCAGCTCTGTTCAAAGTAAAAC-3'). The sequence of the
IL-1HY2 coding region was confirmed by sequencing. The NdeI/HindIII restriction fragment containing
IL-1HY2 was isolated and inserted between the NdeI and
HindIII sites of the bacterial expression vector pRSET B
(Invitrogen). The resulting plasmid, pRSET IL-1HY2, was transformed
into Escherichia coli strain BL21(DE3)pLysS (Invitrogen).
IL-1HY2 protein expression was induced by
isopropyl-1-thio-
-D-galactopyranoside, and cells
expressing IL-1HY2 were harvested by centrifugation at 10, 000 × g for 30 min. The cells were then lysed in 10 volumes of
lysis buffer (20 mM Tris, pH 8.0, 1 mM EDTA) by
passing through an Avestin homogenizer. IL-1HY2 present in the
supernatant was purified by a combination of Q-Sepharose anion
exchange, phenyl-Sepharose hydrophobic interaction, and DEAE-Sepharose
anion exchange chromatography (AP Biotech). The IL-1HY2 protein was
95% pure as determined by SDS-polyacrylamide gel electrophoresis and
Coomassie staining. The dissociation equilibrium constants
(KD) of IL-1HY2, IL-1ra, and IL-1
binding to
soluble IL-1 receptor type I (sIL-1RI) were measured using the IAsys
affinity sensor (Labsystems Affinity Sensors, Franklin, MA) according
to the manufacturer's instructions and as described (14, 15). Purified
recombinant IL-1HY2 protein, recombinant IL-1ra, and IL-1
proteins
(R & D Systems) were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin
(Pierce) according to the manufacturer's instructions. The
biotinylated proteins (1.2 µM) were immobilized onto the
avidin-layered biotin-coated cuvette surface. After ligand immobilization, sIL-1RI (R & D Systems) at various concentrations (70-500 nM) was added to the cuvette. Protein binding
kinetics was measured, and the KD of each protein to
the IL-1 receptor was obtained using the software Fastfit (Labsystems
Affinity Sensors) (14, 15).
Generation of Antibodies and
Immunohistochemistry--
Anti-IL-1HY2 polyclonal antibodies were
raised in rabbit against a synthetic peptide (CTLPNRGLDRTKVP) specific
to the IL-1HY2 protein. The antibodies specific to the peptide were
subsequently isolated by affinity purification using standard
procedures (16). The anti-IL-1HY2 antibodies did not cross-react with
other IL-1 family members, including IL-1ra, IL-1
, and IL-1HY1, in a
Western blot analysis (data not shown). The anti-CD20 monoclonal
antibody was purchased from DAKO (Carpenteria, CA), and the anti-Ki67
was obtained from Coulter Immunotech (Miami, FL). Immunohistochemistry was performed at QualTek Molecular Systems, Inc. (Santa Barbara, CA)
using a procedure described previously (17). Briefly, sections of adult
human tonsil and skin were reacted with the primary anti-IL-1HY2 antibodies that were detected by a biotinylated secondary antibody followed by streptavidin-AP. Fast Red was used as the chromogen (red),
and the slides were counter stained with hematoxylin (blue nuclear
stain). In the negative control staining, slides were processed without
adding the primary antibody. For the double labeling
immunohistochemistry, the second primary antibody, the anti-CD20
monoclonal antibody, or the anti-Ki67 monoclonal antibody, was detected
using a biotinylated secondary antibody followed by
streptavidin-horseradish peroxidase. Diaminobenzidine (DAB) was
used as the chromogen (brown).
Three-dimensional Structure Modeling--
The
GeneAtlasTM software package (Molecular Simulations Inc.,
San Diego, CA) was used to generate three-dimensional structural models
for IL-1HY2. The three-dimensional structural models of the IL-1HY2
protein were predicted based on a search of 4250 nonredundant Protein
Data Bank structures using the multiple alignment sequence profile-based searching method (PSI-BLAST) (18), an automated sequence
and structure searching procedure (High Throughput Modeling) (19), and
a fold recognition method (SeqFold) (20). The known structures of
IL-1ra (21) and IL-1
(22) (1i1b for IL-1
and 1irp, 1ilr, and 1ilt
for IL-1ra) that were the best fit structures were used as templates.
 |
RESULTS |
To identify additional IL-1 family member genes, BLAST and motif
algorithms were used to search an expressed human gene sequence data
base generated using screening by hybridization technology (10,
11). A cDNA sequence from a human fetal skin library was identified
that encodes a polypeptide with homology to the IL-1ra protein.
Sequencing of the cDNA clone and subsequent 5' rapid amplification
of cDNA ends analysis led to the identification of a full-length
coding sequence designated as IL-1HY2. The nucleotide and
the deduced protein sequences are shown in Fig.
1. Blast search analysis indicated that
this nucleotide sequence had not been previously reported in any public
data bases. The IL-1HY2 cDNA contains an open reading frame
corresponding to a 152-amino acid residue protein with a predicted
molecular mass of 17 kDa and a predicted isoelectric point of
4.7.

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Fig. 1.
cDNA and deduced amino acid
sequences of human IL-1HY2 (GenBankTM accession
number AF334755). The putative polyadenylation signal is
underlined.
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|
Sequence analysis indicated that the IL-1HY2 protein is highly
homologous to IL-1HY1 (41% similarity) and IL-1ra (37% similarity), whereas it shares lower homology (14-30%) with IL-1
and other IL-1
family proteins (Fig. 2, A and
B). Based on amino acid similarities among the IL-1 family
members, IL-1HY2, IL-1HY1, and IL-1ra may be grouped into a subfamily,
and FIL1
, FIL1
, FIL1
, and IL-1H1 belong to a second subfamily,
whereas IL-1
, IL-1
, and IL-18 fall to a third subfamily (Fig.
2C).

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Fig. 2.
Sequence comparison of IL-1HY2 and other IL-1
family proteins. A, amino acid sequence alignment of
human IL-1HY2, intracellular IL-1ra, IL-1HY1, and IL-1 (mature
form). Sequence alignment was obtained using the ClustalW algorithm of
the Lasergene software (DNASTAR Inc., Madison, WI). The boxed
areas indicate matching residues. The vertical arrows
indicate intron positions. B, sequence pairwise comparison
of IL-1 family members. The numbers represent the percentage
of sequence similarity determined by the J. Hein method with
PAM250 residue weight table in the Lasergene software.
C, phylogenetic analysis of the IL-1 family. The inferred
phylogenetic tree was generated based on degree of amino acid sequence
similarity shown in B. Accession numbers in the public data
bases for proteins used in this figure are as follows: IL-1 , P01583
(mature protein, amino acid residues 113-271); IL-1 , P01584 (mature
protein, amino acid residues 117-269); intracellular IL-1ra, AAB92268;
IL-18, BAA08706 (mature protein, amino acid residues 37-193); IL-1HY1,
AF186094; FIL-1 , AF201831; FIL-1 , AF201833; FIL-1 , AF201832;
and IL-1H1, AF200492.
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The IL-1HY2 locus was mapped to human chromosome 2 using the
NIGMS human/rodent somatic cell hybrid mapping panel (12). The Stanford
G3 human/hamster radiation hybrid panel (13) was used to further
localize the IL-1HY2 gene to a region on human chromosome
2q14 with a distance of 7 centiRays from the marker SHGC-7020
and a LOD score of 10.58. Interestingly, IL-1
(marker SHGC-10703), IL-1
(marker SHGC-11912),
IL-1ra (marker WI7030), IL-1HY1 (marker
SHGC-7020), and FIL1
, FIL1
, and
FIL1
were also mapped to a similar location on chromosome
2 (5, 7-9, 23, 24).
The genomic organization of the IL-1HY2 gene was determined
by sequencing of the human BAC clone containing the IL-1HY2
gene (see "Experimental Procedures"). Like the other IL-1 family
members, the IL-1HY2 gene consists of four coding exons
(Fig. 3). The structure of the
IL-1HY2 gene is remarkably similar to that of the other IL-1
family genes, with a conserved pattern of intron placement (Fig.
2A).

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Fig. 3.
Organization of the human IL-1HY2
gene (GenBankTM accession number AF334756).
Horizontal lines represent introns, and boxes
represent exons. Black areas of the boxes
represent coding sequences. Sizes of exons and introns are indicated in
nucleotides (nt).
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To determine the expression pattern of the IL-1HY2 gene, a
panel of cDNA libraries derived from various human tissues was analyzed by a semi-quantitative PCR using primers specific to IL-1HY2.
The IL-1HY2 transcripts were expressed in fetal skin and at a lower
level in spleen (Fig. 4A). To
determine whether IL-1HY2 protein is secreted in mammalian cells, an
IL-1HY2 expression construct was used to transfect CHO cells. The
recombinant IL-1HY2 protein was detected in both the cell lysate and
conditioned medium from transiently transfected CHO cells (Fig.
4B).

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Fig. 4.
IL-1HY2 expression in human tissues and
secretion of the IL-1HY2 protein in transiently transfected CHO
cells. A, IL-1HY2 expression analysis. Plasmid DNA from
a panel of cDNA libraries was analyzed by PCR using primers
specific to IL-1HY2 and -actin. The cDNAs were derived from the
following human tissues: heart (lane 1); brain (lane
2); liver (lane 3); lung (lane 4); spleen
(lane 5); ovary (lane 6); testis (lane
7); kidney (lane8 ); placenta (lane 9);
fetal brain (lane 10); fetal skin (lane 11);
fetal lung (lane 12); and fetal liver (lane 13).
B, secretion of IL-1HY2 protein in transiently transfected
CHO cells. CHO cells were transfected transiently with the IL-1HY2
expression construct. Proteins from the cell lysate and culture medium
(10-fold concentrated) were analyzed by Western blot using polyclonal
antibodies against the IL-1HY2 protein. A vector without the IL-1HY2
cDNA was used as a control in the transfection of CHO cells.
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Because the IL-1HY2 protein, which belongs to the IL-1 family, is
secreted in mammalian cells, the recombinant IL-1HY2 protein was tested
for binding to the sIL-1RI using the IAsys affinity sensor.
Biotinylated recombinant IL-1HY2, IL-1ra, or IL-1
protein was
immobilized on the avidin-layered biotin-coated cuvette surface, and
their dissociation equilibrium constants (KD) with sIL-1RI were measured (see "Experimental Procedures"). The
recombinant IL-1HY2 protein bound sIL-1RI, although the binding
affinity of IL-1HY2 was lower (higher KD) than those
of IL-1ra and IL-1
(Table I).
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Table I
Dissociation equilibrium constants (KD) of IL-1HY2, IL-1ra,
and IL-1 binding to sIL-1RI
The dissociation equilibrium constant (KD) was
measured using the IAsys affinity sensor (Labsystems Affinity Sensors)
as described (14, 15). Data from a representative experiment are
presented.
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To analyze IL-1HY2 protein expression in the human skin, polyclonal
antibodies raised against a peptide specific to IL-1HY2 were used in
the immunohistochemistry study (Fig. 5).
IL-1HY2 protein was expressed mostly in the basal epithelia of the
human skin (Fig. 5B). To identify the cell types in immune
tissues that express the IL-1HY2 protein, immunohistochemistry was
performed on the tonsil (Fig. 6). From
the staining data, the IL-1HY2 protein was expressed in the germinal
center of the tonsil (Fig. 6B). Using double labeling
immunostaining, the IL-1HY2 protein was detected in a subset of B cells
(CD-20-positive) (Fig. 6C), most of which were proliferating
(Ki67-positive) cells (Fig. 6D). Furthermore, the
IL-1HY2-positive cells did not react with the anti-CD45RO (T cell
marker) antibody or the anti-CD14 (monocyte marker) antibody (data not
shown), suggesting that the IL-1HY2 protein was not expressed in T
cells or monocytes in the tonsil.

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Fig. 5.
Analysis of IL-1HY2 protein expression in
human skin by immunohistochemistry. Sections of the human skin
were processed without primary antibodies (A) or with
affinity purified polyclonal antibodies specific to the IL-1HY2
polypeptide (B). A biotinylated secondary antibody was used
followed by streptavidin-AP. The anti-IL-1HY2 antibodies were
detected using Fast Red as the chromogen (red). Slides were
counter-stained with hematoxylin (blue nuclear stain).
Pictures are shown at 150× magnification.
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Fig. 6.
Cell type-specific expression of the IL-1HY2
protein in the human tonsil. Sections of the human tonsil were
reacted without primary antibodies (A) or with affinity
purified polyclonal antibodies specific to the IL-1HY2 polypeptide
(B) as described in the legend for Fig. 5. For double
labeling of the tissues, anti-CD20 antibody was used as a second
primary antibody in addition to the anti-IL-1HY2 antibodies
(C) or the anti-IL-1HY2 and anti-Ki67 antibodies
(D) were used as primary antibodies. The second primary
antibody (anti-CD20 antibody or anti-Ki67 antibody) was detected using
a biotinylated secondary antibody followed by
streptavidin-horseradish peroxidase. Diaminobenzidine
(DAB) was used as the chromogen (brown). Sections
shown represent the edges of a germinal center within a tonsil.
Pictures are shown at 400× magnification.
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 |
DISCUSSION |
Recently, five new IL-1 family members, including
IL-1HY1 (5), FIL1
, FIL1
, and
FIL1
(7), and IL-1H1 (8), were identified. In
this report, we describe the discovery of a novel IL-1 family gene,
IL-1HY2, expanding the IL-1 family to include a total of 10 family members (IL-1
, IL-1
,
IL-1ra, IL-18, IL-1HY1, FIL1
, FIL1
, FIL1
,
IL-1H1, and IL-1HY2). It would appear that IL-1HY2 was derived from a duplication event from an
ancestral gene common to the other IL-1 family members. This notion is
evidenced by significant amino acid sequence homologies (19-41%) that
the IL-1HY2 protein shares with the other IL-1 family members (Fig. 2,
A and B) and by the high degree of conservation
in the genomic organization of the IL-1HY2 gene with the
other family member genes (Figs. 1 and 3). Based on the amino acid
sequence homologies, IL-1HY2, IL-1HY1, and IL-1ra appeared to form a
subfamily that evolved from a common ancestor (Fig. 2C).
The three-dimensional structural model of the IL-1HY2 protein was
predicted based on a search of 4250 nonredundant Protein Data Bank
structures. The known crystal structure of IL-1ra (21), which was one
of the best fit structures, was used as a template (Fig.
7). The IL-1HY2 structural model displays
a 12-
-stranded trefoil structure and is highly similar to the IL-1ra
crystal structure (Fig. 7). The structural model of IL-1HY2 predicted based on the IL-1
crystal structure (22) also shares similarity to
the IL-1
structure (data not shown). The similarity between the
IL-1HY2 structural models and the IL-1ra and IL-1
structures provides more evidence that IL-1HY2 belongs to the IL-1 family.

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Fig. 7.
Comparison of the three-dimensional
structural model of IL-1HY2 with the crystal structure of IL-1ra.
The IL-1HY2 structural model was generated using the
GeneAtlasTM software package (Molecular Simulations Inc.,
San Diego, CA). A and C, the crystal structure of
IL-1ra (blue) and the predicted 12- -stranded trefoil
structural model of IL-1HY2 (yellow) based on the IL-1ra
template, respectively. Both the crystal structure and the model are
viewed down the -barrel axis. B, the superimposed crystal
structure and the IL-1HY2 structural model.
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The recombinant IL-1HY2 proteins expressed in mammalian (CHO) cells had
two forms, a major form at 25 kDa and a minor form at 17 kDa (Fig.
4B), whereas the predicted molecular mass based on the amino
acid sequence of IL-1HY2 is 17 kDa. The major form of the IL-1HY2 may
be a result of post-translational modifications of the protein.
However, the IL-1HY2 protein lacks any N-glycosylation consensus sites. Neither N-glycosylation nor
O-glycosylation on the recombinant IL-1HY2 protein expressed
in CHO cells was detected using peptide N-glycosidase F and
O-glycosidase deglycosylation analysis (data not shown).
Thus, the difference between the apparent molecular mass and the
predicted molecular mass of IL-1HY2 may be due to other
post-translational modifications, such as phosphorylation.
Although IL-1HY2 lacks an obvious signal peptide, we have demonstrated
that the recombinant IL-1HY2 protein is secreted from CHO cells (Fig.
3B), suggesting that IL-1HY2 may act as a secreted ligand.
Other IL-1 family proteins, including IL-1
and IL-18, also lack a
classical signal peptide and are still secreted. The secretion of
IL-1
and IL-18 involves cleavage by the IL-1
converting enzyme
(25-27). The IL-1HY2 protein does not contain an apparent consensus
site for the IL-1
converting enzyme cleavage. Thus, secretion of the
IL-1HY2 protein may involve an alternative secretory mechanism.
IL-1HY2 was shown to bind sIL-1RI, although it does not bind as avidly
as IL-1
and IL-1ra to sIL-1RI (Table I). The KD values of IL-1ra and IL-1
binding to sIL-1RI presented here are higher than those reported by other groups (28, 29). This may reflect
the different methodology and instrumentation (IAsys affinity sensor)
that we used. Thus, the binding affinities of IL-1HY2 can only be
compared with those of IL-1ra and IL-1
determined using the same
conditions. The affinity of IL-1HY2 binding to the IL-1 receptor was
determined using the soluble form of the receptor. It has been shown
that IL-1 receptor accessory protein plays a role in enhancing IL-1
receptor-ligand binding affinities and signaling activities (30, 31).
Thus, the IL-1 receptor accessory protein or another accessory protein
may affect the binding affinity of IL-1HY2 to the IL-1 receptor. The
fact that IL-1HY2 binds to sIL-1RI suggests that IL-1HY2 may play a
role in regulating the IL-1 receptor function.
The data presented in the current report indicate that IL-1HY2 is
expressed in skin and activated B cells of the human tonsil. The
tissue-specific expression pattern of IL-1HY2 suggests that it may be
involved in the regulation of normal immune responses and inflammatory pathophysiology.
 |
ACKNOWLEDGEMENTS |
We thank Matthew Arterburn, Mark Dickson, and
the sequencing group at Hyseq Inc. for sequencing analysis, Daryl Love,
John Childs, and Henryk Cudny for bacterial recombinant protein
production and purification, and Page Erickson and Steve Bernstein at
QualTek Molecular Systems, Inc. (Santa Barbara, CA) for
immunohistochemistry. We also thank Drs. Fabio Rupp and Julio J. Mulero
for critical reading of the manuscript and Phil Santamaria for help
with preparation of graphics.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF334755 and AF334756.
Present address: Center for Biomolecular Science and Engineering,
University of California, Santa Cruz, CA 95064.
§
To whom correspondence should be addressed: Functional Genomics
Dept., Hyseq Inc., 670 Almanor Ave., Sunnyvale, CA 94086. Tel.:
408-524-8100; Fax: 408-524-8141; E-mail: ford@sbh.com.
Published, JBC Papers in Press, March 14, 2001, DOI 10.1074/jbc.M010095200
 |
ABBREVIATIONS |
The abbreviations used are:
IL, interleukin;
IL-1ra, IL-1 receptor antagonist;
sIL-1RI, soluble IL-1 receptor type
I;
PCR, polymerase chain reaction;
CHO, Chinese hamster ovary..
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