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MediCity Research Laboratory, University of Turku, and National Public Health Institute, Turku, Finland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human vascular adhesion protein-1 (VAP-1) is an important endothelial cell adhesion molecule involved in the binding of lymphocytes to endothelium (6). In vivo human VAP-1 (hVAP-1) is a 170- to 180-kDa dimer consisting of two identical subunits of 90 kDa (7). It is expressed mainly in the HEV of peripheral lymph node (PLN)-type lymphatic tissues (6), and the surface expression of VAP-1 is up-regulated after prolonged chronic inflammation in different tissues, such as gut, tonsil, skin, and synovium (8, 9). Based on the reactivity with the original anti-VAP-1 mAb 1B2, VAP-1, with a slightly different molecular mass, can also be found in smooth muscle cells and follicular dendritic cells, although its function on these cells is unknown.
VAP-1 is distinct from the peripheral lymph node addressin (PNAd), defined by mAb MECA-79 (10), although both VAP-1 and PNAd can mediate lymphocyte binding to PLN under shear conditions. However, in contrast to PNAd, VAP-1 can operate in an L-selectin-independent manner and support the binding of L-selectin-negative lymphocytes to the endothelium (11). Studies of tonsillar VAP-1 using digestion with specific glycosidases have shown that VAP-1 is a sialoglycoprotein, probably containing both N- and O-linked sugars with abundant sialic acid residues. It has also been shown that sialic acids are required for the adhesive properties of VAP-1, since the desialylated molecule can no longer support lymphocyte binding in the frozen section assay (7).
We have recently isolated the cDNA encoding the human VAP-1 and shown that it encodes a novel functional adhesion molecule (38). The hVAP-1 cDNA encodes a type II transmembrane protein of 84.6 kDa with six potential N-glycosylation sites and three potential O-glycosylation sites in the long extracellular domain. The hVAP-1 cDNA transfected Ax endothelial cells that express VAP-1 on their surface bind lymphocytes, and the binding can be inhibited with anti-hVAP-1 mAbs. The hVAP-1 protein sequence has no similarity to any known adhesion molecules, but it has significant identity to a family of enzymes called the copper-containing amine oxidases.
In this paper we describe the isolation of mouse VAP-1 cDNA and production of a mAb against this mouse molecule. The isolated cDNA encodes a previously unknown mouse molecule with highly significant identity to human VAP-1 and also to a family of enzymes called copper-containing amine oxidases. We also show that this mouse molecule possesses enzyme activity against monoamines and characterize its m.w. and tissue distribution with the new anti-mVAP-1 mAb. Taken together, these results suggest that we have cloned mouse VAP-1, which will allow us to study in vivo the function and significance of VAP-1 in lymphocyte recirculation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Total RNA from BALB/c mouse kidney was prepared using an Ultraspec kit (Biotecx, Houston, TX), and a mVAP-1 PCR fragment was amplified using partially degenerate oligonucleotide primers PB3 and PB5 (KEBO Laboratory, Espoo, Finland): PB3 TTTTGTGTGTTYGARCARAAY (designed from the hVAP-1 protein sequence FCVFEQN; residues 429–435 of the predicted hVAP-1 protein sequence) and PB5 GTTRGGAATRTCYTCWGCRTG (designed from the hVAP-1 protein sequence HAEDIPN; residues 687–693, respectively). The reaction conditions in PCR were 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min, repeated for 30 cycles. The amplified 800-bp fragment was then blunt end cloned into pUC18 vector using a SureClone kit (Pharmacia, Uppsala, Sweden). The cloned DNA fragment was sequenced using a Sequenase version 2.0 kit (U.S. Biochemical Corp., Cleveland, OH) and the sequencing service facilities of the Department of Medical Genetics, University of Turku (Turku, Finland). Sequence analysis and database comparisons were performed using the Wisconsin Package version 8.1-UNIX of the Computer Group (Madison, WI).
Approximately 1 x 106 recombinant 
phage from
a mouse heart 5' STRETCH PLUS cDNA library in the gt10 cloning
vector (Clontech Laboratories, Palo Alto, CA) were screened with the
reverse transcriptase-PCR-amplified mVAP-1 PCR fragment from mouse
kidney mRNA. The fragment was labeled using an Amersham Multiprime DNA
labeling kit and [
-32P]dCTP at about 3000 Ci/mmol
(Amersham International, Aylesbury, U.K.). Approximately 100,000
plaques/plate were transferred to Hybond-N nylon filters (Amersham),
and duplicate filters were hybridized at 65°C in 5x SSC, 5x
Denhardt’s reagent, 0.5% SDS, and 0.5 mg/ml denatured, sheared salmon
sperm DNA. The filters were washed twice for 30 min each time at 65°C
in 0.1 x SSC and 0.1% SDS and autoradiographed with intensifying
screens (Biomax MS film, Eastman Kodak, Rochester, NY). One positive
clone was identified, which was subjected to secondary screening, from
which a single plaque was purified, and the insert of this clone was
subcloned into EcoRI-digested pUC18 for sequencing. Plaque
hybridization, phage purification, Escherichia coli
transformation, and plasmid purifications were all performed according
to standard molecular biology protocols (12).
Northern blot analysis
A mouse multiple tissue Northern blot was obtained from Clontech Laboratories (Palo Alto, CA) and was hybridized according to the manufacturer’s instructions at 65°C using the PCR-amplified 800-bp mVAP-1 PCR fragment as a probe.
Abs and immunohistochemistry
An anti-mVAP-1 mAb was produced by immunizing rats with recombinant mVAP-1. In brief, soluble mVAP-1 fusion protein was produced by amplifying a 117-bp long C-terminal part of the mVAP-1 cDNA with PCR primers MVAPGST8; GTC AGG ATC CGG GAG GGC CAG GAT G (designed from the mVAP-1 protein sequence REGQDA; residues 726–731 of the predicted protein sequence) and MVAPGST9; TCC CGA ATT CTG TCT CTG TAA GCA AAG C (designed from the protein sequence GFAYRDN; residues 759–765, respectively). Reaction conditions in the amplification were 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min for 30 cycles. The amplified fragment was digested with BamHI and EcoRI and ligated into BamHI- and EcoRI-digested pGEX3X vector (glutathione-S-transferase (GST) gene fusion vector, Pharmacia). A portion of the fusion protein was run on SDS-PAGE gel and stained with Coomassie blue to confirm the purity and solubility of the resulting GST-mVAP-1 fusion protein. Finally, the fusion protein was mixed with IFA and used to immunize specific pathogen-free Sprague-Dawley rats. After three s.c. injections into the foodpads at 1-wk intervals, the rats were sacrificed, and the popliteal lymph nodes were collected. The isolated lymphocytes were then fused with nonsecreting Sp2/0 myeloma cells using standard procedures. The resulting hybridoma supernatants were tested by immunoperoxidase staining of acetone-fixed cryostat sections obtained from specific pathogen-free BALB/c mice gut. The sections were incubated with primary Abs (100 µl of hybridoma supernatant from mVAP-1 fusions or 100 µl of supernatant from a hybridoma producing a negative control mAb, Hermes-1, which is a rat mAb against human CD44) (13) for 120 min, washed twice in PBS, and incubated for 30 min with peroxidase-conjugated goat anti-rat Ig (Dako, Glostrup, Denmark) containing 5% normal mouse serum. The reaction was developed for 5 min using 3',3'-diaminobenzidine hydrochloride in PBS containing 0.03% hydrogen peroxide as a chromogen. After the stainings, the sections were counterstained with hematoxylin. One hybridoma supernatant (TK10–79) positively stained the sections and was subcloned twice by limiting dilution before use in additional experiments.
Transfections and cell culture
For transfections, the mVAP-1 cDNA fragment covering 1 to 3101
bp was isolated from pUC18 after EcoRI and XbaI
digestions and subcloned into the pcDNA3 expression vector (Invitrogen,
San Diego, CA). CHO cells were used as hosts in transfections to
express the mVAP-1 protein, and they were grown in -MEM (Life
Technologies, Paisley, U.K.) plus CHO nucleosides supplemented with
20% FCS, 2 mM glutamine (Biological Industries, Beit Haemek, Israel),
128 U/ml penicillin, and 128 µg/ml streptomycin. Electroporation was
performed using a Bio-Rad Gene Pulser apparatus (Richmond, CA; 0.3 kV,
960 µF, 0.4-cm cuvette in RPMI plus 1 mM sodium pyruvate, 2 mM
L-glutamine without serum, and 20 µg of expression
plasmid). These stably transfected cells were selected by culturing
them in the presence of 0.5 mg/ml geneticin (Life Technologies, Grand
Island, NY).
Immunoblotting
mVAP-1 CHO transfectants were scraped from cell culture flasks and lysed with lysis buffer (150 mM NaCl, 10 mM Tris base (pH 7.2), 1.5 mM MgCl2, 1% Nonidet P-40, 1% aprotinin, and 1 mM PMSF). After 2 h at 7°C, the lysates were centrifuged (13,000 rpm, 30 min, 7°C), and the supernatants were collected. Noninflamed mouse gut samples from BALB/c mice were dissected free from the lamina propria. The smooth muscle wall was minced into small pieces, lysed 2 h in the lysis buffer at 7°C, and centrifuged, and the supernatants were collected. Twenty-five microliters of the gut smooth muscle lysate supernatant or CHO cell lysate supernatant was mixed with an equal volume of Laemmli’s sample buffer for electrophoresis under nonreducing conditions. Before loading on a 5 to 12.5% SDS-PAGE gel, the samples were boiled for 5 min at 95°C. The resolved proteins were transferred onto nitrocellulose sheets (Hybond-ECL, Amersham) by a Hoefer electroblotter (San Francisco, CA). Nitrocellulose strips were stained using an enhanced chemiluminescence detection kit (Amersham) according to the manufacturer’s recommendations. Briefly, blocking was performed with PBS containing 10% nonfat milk powder and 0.3% Tween-20 for 1 h, and a 1/20 dilution of the hybridoma TK10–79 supernatant was used as a primary Ab. The mAb Hermes-1 was used as a negative control Ab. Peroxidase-conjugated goat anti-rat Ig containing 5% normal mouse serum was used in the second stage of staining.
Amine oxidase assays
The mVAP-1-transfected and mock (pcDNA3 vector) transfected CHO
cells (typically 10–15 x 106) were scraped into
10 ml of PBS and centrifuged at 4°C. The cell pellet was washed twice
with PBS and finally resuspended in 1 ml of PBS before sonication on
ice at medium power (Braun sonicator, Melsungen, Germany). Sonicated
lysates were used in the assay immediately or after storage at
-20°C. All enzyme reactions were performed with or without heating
the cell lysates for 5 min at 95°C before addition of the substrate.
The enzyme activity assay protocol was modified from the method
described by D’Agostino (14). Briefly, in benzylamine assay, 100 µl
of the cell lysate was mixed with 300 µl of 100 mM NaPO4
and 100 µl of the substrate, and in the putrescine assay, 100 µl of
the cell lysate was mixed with 1.7 ml of 100 mM NaPO4, 200
µl of 400 mM acetaldehyde, and 100 µl of the substrate.
[14C]benzylamine hydrochloride (0.2 µCi; Amersham) in
100 mM NaPO4 containing 0.8 mM unlabeled benzylamine
(Sigma, St. Louis, MO) or 0.1 µCi of [14C]putrescine
dihydrochloride (Amersham) plus 0.04 mM unlabeled putrescine (Sigma)
were used as substrate in these reactions. One milligram of porcine
kidney diamine oxidase (Sigma) was used as a positive control for
diamine oxidase activity, and 0.5 mg of bovine plasma monoamine oxidase
was used as a control source for monoamine oxidase activity. The
reactions were incubated at 37°C for 60 min before double extraction
of the labeled aldehyde reaction products in 700 µl of toluene-based
scintillation mixture containing toluene and 0.35 g/l diphenyloxazole.
The extractions were measured for 14C by a liquid
scintillation counter (15). Hydroxylamine hydrochloride (5 µM; Sigma)
and semicarbazide hydrochloride (0.1 mM; Sigma) were used as specific
inhibitors (16) and incubated for 10 min with 100 µl of the cell
lysate before addition of the substrate. Total protein concentration
measurements were performed using the Bradford method with bovine
-globulin as a standard. The results are presented as picomoles of
substrate used per minute per milligram of protein.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Based on the hVAP-1 sequence, several degenerate oligonucleotide
primers were designed to be used in reverse transcriptase-PCR
experiments with mRNA isolated from different mouse tissues. A single
800-bp fragment of the expected size was amplified from mouse kidney
mRNA with the degenerate oligonucleotides PB3 and PB5 as primers, and
this specific fragment was cloned for sequencing. The sequence revealed
that the amplified 800-bp fragment had 80% amino acid identity to
hVAP-1, indicating that the amplified fragment could represent mVAP-1.
PCR screening of different mouse libraries showed that a mouse heart
cDNA library was positive with the same primer pair, so this library
was selected for screening with the PCR-amplified mVAP-1 fragment as a
probe to search for the full-length mVAP-1 cDNA. One phage with a large
4.4-kb insert was isolated, cloned, and sequenced, and the preliminary
sequence revealed that the isolated cDNA contained the whole 800-bp PCR
fragment sequence. Thus, the whole isolated insert was sequenced in two
directions. The insert had an ATG starting codon 350 bp down from the
5' end of the cDNA and was followed by one continuous open reading
frame of 2298 bp (Fig. 1
). A 5'
untranslated region of 349 bp and a 3' untranslated region of 1830 bp
following the TGA stop codon were present in the isolated clone. At the
3' end of the clone, a polyadenylation signal and a poly(A) tail were
found, suggesting that the isolated clone also included the whole 3'
end of the cDNA encoding mVAP-1.
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The open reading frame encoded a 765-amino acid protein with a
predicted molecular size of 84.5 kDa (Fig. 1
). The mVAP-1 protein has
six potential N-glycosylation sites and six potential
O-glycosylation sites (according to the
O-glycosylation site prediction server, NetOglyc@cbs.dtu.dk
(17)). The predicted amino acid sequence revealed that the identity of
mVAP-1 to hVAP-1 is 83%, distributed evenly throughout the molecules.
A search of the most recent releases from SwissProt and GenEMBL
sequence databanks revealed that mVAP-1 has no significant identity to
any other adhesion molecule or mouse protein, but it has, like hVAP-1,
significant identity to a family of enzymes called copper-containing
amine oxidases.
Amine oxidases are enzymes classified into two main classes on the
basis of the chemical nature of their cofactors (16, 18): the flavin
adenine dinucleotide (FAD)-containing amine oxidases and the
copper-containing amine oxidases. The FAD-containing amine oxidases
include both the A and B forms of the widely studied outer
mitochondrial membrane monoamine oxidases, which possess activity not
only against primary amines but also against secondary and tertiary
amines (19). In addition to the differences in their cofactors, the
copper-containing amine oxidases are distinct from the FAD-containing
amine oxidases with regard to their location inside the cell and their
substrate specificity. A multiple alignment of mVAP-1, hVAP-1, and five
other cloned mammalian members of the copper-containing amine oxidases
is shown in Figure 2. The identity of
mVAP-1 to these molecules varied between 42 and 95%. The highest
identity (95%) was found to the recently published partial cDNA
sequence of a rat membrane-bound amine oxidase (20), which together
with hVAP-1 comprises the first identified transmembrane protein
members of this family. Figure 2 shows also that the hydrophobic amino
acids present in the rat (residues 6–26) and human (residues 5–27)
membrane-spanning regions are 100% identical between mouse and rat
molecules and highly conserved between mouse and human VAP-1. In
contrast, the putative transmembrane sequence in the mouse has no
homology to other members of the copper-containing amine oxidase family
(for example bovine serum amine oxidase (BSAO)), which are secreted
proteins. This indicates that mVAP-1 probably has a transmembrane
domain close to the N-terminus of the molecule and that it belongs to
the membrane-bound glycoprotein group of the copper-containing amine
oxidase family.
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In Northern blot analysis of mRNA from BALB/c mice using the
800-bp mVAP-1 PCR fragment as a probe, a single 4.4-kb band was found
to hybridize from different tissues (Fig. 3). No other mRNA species were detected
even after prolonged autoradiography (data not shown). The expression
levels varied; they were strong in heart, lung, skeletal muscle,
kidney, and testis. In liver, brain, and spleen only low amounts of
mVAP-1 mRNA were detected in longer exposures. The expression pattern
of mVAP-1 mRNA is very similar to that of hVAP-1, except that in mouse
brain a weak mRNA message was detected during the prolonged exposure,
whereas human brain did not express any detectable hVAP-1 mRNA in
Northern blot analysis (data not shown).
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The fact that mVAP-1 is the first cloned mouse molecule with
significant identity to proteins belonging to the amine oxidase family
(16) led us to test whether mVAP-1 possesses enzyme activity against
different amines. In enzyme assays, mVAP-1-transfected CHO cell lysates
were assayed with benzylamine (a monoamine) or putrescine (a diamine)
as a substrate. In these experiments mVAP-1 showed a clear activity
against benzylamine but not against putrescine (Table I), and the activity could be destroyed
by heating the cell lysates for 5 min at 95°C before addition of the
substrate. Mock transfected CHO cell lysates did not show activity
against any substrates. Porcine kidney diamine oxidase (for diamine
oxidase activity) and bovine plasma monoamine oxidase (for monoamine
oxidase activity) were used as positive controls (18, 21) against
putrescine and benzylamine, respectively, and the activities of these
controls were abolished by heating. To test whether mVAP-1 activity
against benzylamine could be inhibited by semicarbazide or
hydroxylamine, two known inhibitors of copper-containing amine oxidases
(16), these inhibitors were incubated separately with the
mVAP-1-transfected CHO cell lysates before addition of the substrate.
In the presence of 0.1 mM semicarbazide or 5 µM hydroxylamine, mVAP-1
showed no activity against benzylamine (Table II). In conclusion, mVAP-1 has a clear
activity against benzylamine but not against putrescine (a diamine),
and the activity is inhibitable with the known copper-containing amine
oxidase inhibitors.
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To study the expression of mVAP-1 and the molecular size of mVAP-1, an anti-mVAP-1 mAb was produced. First, a C-terminal 117-bp fragment of the mVAP-1 cDNA (coding for amino acids 726–765 in the mVAP-1 protein sequence) was amplified with PCR and cloned into the pGEX3X vector to produce a GST-mVAP-1 fusion protein. After isopropylthiogalactoside induction and purification of the resulting recombinant protein from E. coli, part of the sample was run on an SDS-PAGE gel to test its purity and solubility. Because the resulting recombinant protein was soluble and of the expected size, the rest of this material was used to immunize rats from which popliteal lymph node lymphocytes were collected after 3 wk and fused with Sp2/0 mouse myeloma cells. The resulting hybridoma supernatants were screened by immunohistologic staining of frozen tissue sections. Based on the staining reactivity of all anti-hVAP-1 mAbs with smooth muscle in addition to endothelial cells, we decided to test the hybridomas on mouse gut sections. One of the hybridoma supernatants positively stained these gut sections, while the negative controls showed no reactivity, and the staining pattern resembled the that of anti-hVAP-1 mAbs on human gut sections.
To test the specificity of the new mAb, designated TK10-79, lysates
from CHO mVAP-1 transfectants were tested in immunoblotting. Staining
of the filter with mAb TK10-79 revealed specific bands with apparent
molecular masses of 110 and 220 kDa from CHO mVAP-1 transfectants under
nonreducing conditions (Fig. 4,
lane 1), while the mock transfectants (Fig. 4, lanes 2 and 4) and control Ab stainings
were negative. These results confirmed that mAb TK10-79 is specific for
mVAP-1.
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The resulting new anti-mVAP-1 mAb TK10-79 was used in
immunoperoxidase stainings to characterize the expression of mVAP-1 in
mouse PLN and gut. In mouse gut sections, mAb TK10-79 stained smooth
muscle cells (Fig. 5A)
and faintly stained vessels of the lamina propria (Fig. 5B). The staining pattern of mVAP-1 in noninflamed
gut resembles the staining pattern of hVAP-1 in human gut, with hardly
any staining of mucosal HEV in Peyer’s patches. In mouse PLN,
anti-mVAP-1 mAb reactivity was mainly seen on HEV (Fig. 5D) where the initial lymphocyte binding to
endothelium and the transmigration take place. mAb TK10-79 did not
stain any lymphocytes present in PLN. Thus, mVAP-1, like hVAP-1 (8), is
present in both endothelial cells and smooth muscle cells.
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To determine the molecular size of the Ag that the mAb TK10-79
recognizes in tissue sections and to compare the size of this Ag to
that of mVAP-1 CHO transfectants, lysates from mouse gut smooth muscle
and from CHO mVAP-1 transfectants were run in parallel on SDS-PAGE and
used for immunoblotting with the mAb TK10-79. From mouse gut smooth
muscle cell lysate, mAb TK10-79 revealed, under nonreducing conditions,
a band of approximately 110 kDa and a smeared band of around 220 kDa
(Fig. 4, lane 5), indicating that mAb TK10-79 is
detecting in vivo the same size molecule as that from transfectants.
The negative control Ab staining of the gut smooth muscle lysate was
negative. The 220-kDa form of the molecule from both mVAP-1 CHO
transfectants and mouse gut smooth muscle lysates probably represents a
dimeric form, which is a typical feature for hVAP-1 (see Footnote 5)
and a subgroup of membrane-bound amine oxidases
(semicarbazide-sensitive amine oxidases (SSAO), a subgroup of
copper-containing amine oxidases (16, 22)).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The hVAP-1 has been shown to be a transmembrane molecule with a short
four-amino acid intracellular part and a long extracellular domain (see
Footnote 5). Very recently, a new rat transmembrane protein belonging
to the same copper-containing amine oxidase family as hVAP-1 has been
partly cloned, and the predicted protein sequence of this cloned
fragment of the molecule has 95% identity to mVAP-1 and 83% identity
to hVAP-1, indicating that the published partial cDNA is probably
encoding the rat homologue of mouse and human VAP-1 (20). Because the
transmembrane-spanning amino acids among these three molecules (Fig. 2, residues 5–26 in hVAP-1 and 6–26 in rat AO) are highly conserved,
mVAP-1 is probably also a type II transmembrane protein with the
N-terminus of the molecule located intracellularly and the C-terminus
located extracellularly (23). The predicted mVAP-1 amino acid sequence
revealed that mVAP-1 has six potential N-glycosylation and
six putative O-glycosylation sites, and all the potential
glycosylation sites are located in the putative extracellular part of
the molecule. Thus, different oligosaccharides may also play a role in
the function of mVAP-1 as an adhesion molecule, as has been shown for
hVAP-1 (7). The functional importance of these possible oligosaccharide
modifications of mVAP-1 and the adhesion properties of CHO-mVAP-1
transfectants remain to be determined, since the anti-mVAP-1 mAb
TK10-79 is apparently not a function-blocking mAb in adhesion assays.
This is not surprising, since the mAb was raised against a short
peptide sequence of mVAP-1 that was expressed in a bacterial
host.
In immunoblotting experiments the anti-mVAP-1 mAb detected 110- and
220-kDa Ag from mouse gut smooth muscle lysate and from CHO mVAP-1
transfectants. Both 110- and 220-kDa forms of the detected molecule are
larger than the corresponding hVAP-1 bands (
90/170 kDa) detected by
immunoblotting (7). This may be due to the species-specific
glycosylation differences, since the core proteins of the molecules are
almost equal in size, whereas the number of potential glycosylation
sites is even higher in mVAP-1 than in the hVAP-1 protein
core.
The ligands of both hVAP-1 and mVAP-1 are unknown. The predicted
protein sequence of hVAP-1 (see Footnote 5) revealed at residues 726 to
728 an RGD motif that is an important integrin binding site (24)
present in a variety of integrin ligands. At present, the significance
of this RGD motif for the function of hVAP-1 is unknown. Although this
motif is not completely conserved between hVAP-1 and mVAP-1, residues
726 to 728 in mVAP-1 (REG) may also represent a possible integrin
binding site, since a cyclic peptide containing the sequence RE has
been shown to be able to interact with the same or an overlapping
binding site in the 5ß1 integrin as RGD
(25). In principle, in RE, the basic group of arginine is one amino
acid closer to the acidic group of the aspartate than in RGD, making
the sequences REG and RGD closely related in spatial terms (E.
Ruoslahti, unpublished observations).
In addition to the high homology to hVAP-1, the isolated mVAP-1 cDNA
showed significant identity in database comparisons to enzymes
belonging to the family of copper-containing amine oxidases, but no
similarity to FAD-containing amine oxidases, which include monoamine
oxidases A and B. The copper-containing amine oxidases are enzymes that
catalyze the oxidative deamination of various biogenic amines, and they
have widely differing substrate specificities (21). They are composed
of two identical subunits, each containing one copper atom and one
covalently bound carbonyl-containing cofactor, the chemical identity of
which is still controversial. It is not clear whether the cofactor is
the same in each member of the family or whether the function of all
these enzymes is actually copper dependent (26). The cofactor at the
active site of BSAO has been identified as topa-quinone (6-hydroxydopa)
(27). The topa-quinone is formed from a precursor tyrosine that has to
be part of a consensus sequence Asn-Tyr-Asp/Glu to become
post-translationally modified to topa-quinone (28). This active site
consensus sequence is also found in the mVAP-1 sequence in residues 470
to 472. The crystal structure of one copper-containing amine oxidase
(E. coli copper amine oxidase) has been published
(29), and it confirmed the importance of the modified tyrosine residue
in the active site and the existence of three histidines that also have
to be conserved for enzyme activity (27, 30). These three histidines
that coordinate copper binding in the protein core can also be found in
mVAP-1, and they are located in positions 520, 522, and 684 in mVAP-1
cDNA sequence (Fig. 1).
In addition to identification of the structural requirements for the enzyme activity in mVAP-1, we have directly shown that mVAP-1 is enzymatically active. The mVAP-1 cDNA-transfected CHO cell lysates had activity against benzylamine, but not against putrescine, indicating that mVAP-1 has activity at least against monoamines but probably not against diamines. Our results differ from previous reports in which tissue-bound amine oxidase from mouse white adipose tissue homogenate has been shown to possess activity not only against benzylamine but also against acetylputrescine (31). Tissue-specific substrate differences may be one explanation for this detected difference, whereas the most likely explanation is that crude homogenate of the white adipose tissue included more than one amine oxidase that were not separated before the enzyme activity measurements. However, our findings show that the substrate specificity of mVAP-1 closely resembles the specificity of hVAP-1, suggesting that the cloned mouse and human VAP-1 may be close homologues with regard to their substrates in addition to their structural identity. This finding is of great importance, as much species-dependent variation in substrate specificity of amine oxidases has been reported. For example, some amines, such as tyramine and tryptamine, have been shown to be good substrates for the rat enzyme but poor substrates for the human enzyme (32, 33).
The hVAP-1 has been shown to be an ecto-enzyme and to include a quinone cofactor (see Footnote 5). The enzyme activity of hVAP-1 is inhibitable by semicarbazide and hydroxylamine; based on these criteria, hVAP-1 belongs to a subgroup of amine oxidases commonly referred to as SSAO, which are mammalian copper-containing monoamine oxidases found in various mammalian tissues and organs both in the cell membrane and in a soluble form in serum (34). The most detailed information about the substrate specificity and enzyme kinetics of these enzymes is available for BSAO and rat SSAO. We have cloned a novel mouse molecule with activity against monoamines that is inhibitable by carbonyl reagents such as semicarbazide and hydroxylamine. Thus, the cloned mouse molecule most likely belongs to the same SSAO subgroup of amine oxidases as hVAP-1 and represents the first cloned mouse SSAO.
Relatively little is known about the biologic role of SSAO, especially in higher eukaryotes. One problem has been the different substrate specificities, which can limit the value of studies with different laboratory animals in attempts to define the physiologic role of primary amine oxidases in man. The identification and cloning of a mouse molecule with similar enzyme activity to the human homologue will be a valuable tool in future studies on the biologic role of these enzymes. Now it will be possible to investigate in a mouse model whether enzyme activity and lymphocyte adhesion are linked or independent functions.
Although the significance of hVAP-1 as a functionally important endothelial cell adhesion molecule that can mediate early interactions between endothelial cells and lymphocytes in the normal recirculation of lymphocytes as well as in the extravasation of lymphocytes into sites of inflammation has been well established in vitro (6, 7, 8, 9, 11), only limited information on the functional significance of this endothelial cell adhesion molecule in vivo is available. With intravital microscopy, VAP-1 has been shown to be involved in the initial interaction of lymphocytes and endothelial cells in inflamed rabbit mesenterial venules (11). However, a mouse model would be extremely useful for further analyses of the site of VAP-1 in the multistep adhesion cascade and of the significance of VAP-1 in vivo. In this work we have isolated a cDNA encoding mVAP-1, produced an Ab against it, and characterized with this mAb the mouse homologue of hVAP-1. The cloning of the mVAP-1 gene is now in progress, and the targeted disruption of the gene will give valuable information about the role of this adhesion molecule in vivo.
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Acknowledgments |
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Footnotes |
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2 Sequence data have been submitted to the EMBL/GenBank databases under accession number AF054831.
3 Address correspondence and reprint requests to Dr. Petri Bono, MediCity Research Laboratory, University of Turku, Tykistökatu 6A, FIN-20520 Turku, Finland. E-mail address:
4 Abbreviations used in this paper: HEV, high endothelial venule; VAP-1, vascular adhesion protein-1; hVAP-1, human vascular adhesion protein-1; PLN, peripheral lymph node; PNAd, peripheral lymph node addressin; mVAP-1, mouse vascular adhesion protein-1; GST, glutathione S-transferase; FAD, flavin adenine dinucleotide; BSAO, bovine serum amine oxidase; SSAO, semicarbazide-sensitive amine oxidase.
Received for publication November 10, 1997. Accepted for publication January 30, 1998.
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References |
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5ß1 integrin from a phage library. J. Cell Biol. 124:373.This article has been cited by other articles:
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  N. Mercier, M. Osborne-Pellegrin, K. El Hadri, A. Kakou, C. Labat, L. Loufrani, D. Henrion, P. Challande, S. Jalkanen, B. Feve, et al. Carotid arterial stiffness, elastic fibre network and vasoreactivity in semicarbazide-sensitive amine-oxidase null mouse Cardiovasc Res, November 1, 2006; 72(2): 349 - 357. [Abstract] [Full Text] [PDF]
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 T. Martelius, V. Salaspuro, M. Salmi, L. Krogerus, K. Hockerstedt, S. Jalkanen, and I. Lautenschlager Blockade of Vascular Adhesion Protein-1 Inhibits Lymphocyte Infiltration in Rat Liver Allograft Rejection Am. J. Pathol., December 1, 2004; 165(6): 1993 - 2001. [Abstract] [Full Text] [PDF]
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 C. M. Stolen, G. G. Yegutkin, R. Kurkijarvi, P. Bono, K. Alitalo, and S. Jalkanen Origins of Serum Semicarbazide-Sensitive Amine Oxidase Circ. Res., July 9, 2004; 95(1): 50 - 57. [Abstract] [Full Text] [PDF]
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 A. Vega, P. Chacon, J. Monteseirin, R. El Bekay, M. Alvarez, G. Alba, J. Conde, J. Martin-Nieto, F. J. Bedoya, E. Pintado, et al. A new role for monoamine oxidases in the modulation of macrophage-inducible nitric oxide synthase gene expression J. Leukoc. Biol., June 1, 2004; 75(6): 1093 - 1101. [Abstract] [Full Text] [PDF]
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 S.-M. Maula, M. Luukkaa, R. Grenman, D. Jackson, S. Jalkanen, and R. Ristamaki Intratumoral Lymphatics Are Essential for the Metastatic Spread and Prognosis in Squamous Cell Carcinomas of the Head and Neck Region Cancer Res., April 15, 2003; 63(8): 1920 - 1926. [Abstract] [Full Text] [PDF]
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 P. F. Lalor, S. Edwards, G. McNab, M. Salmi, S. Jalkanen, and D. H. Adams Vascular Adhesion Protein-1 Mediates Adhesion and Transmigration of Lymphocytes on Human Hepatic Endothelial Cells J. Immunol., July 15, 2002; 169(2): 983 - 992. [Abstract] [Full Text] [PDF]
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 D. Mason, P. Andre, A. Bensussan, C. Buckley, C. Civin, E. Clark, M. de Haas, S. Goyert, M. Hadam, D. Hart, et al. CD antigens 2002 Blood, May 15, 2002; 99(10): 3877 - 3880. [Full Text] [PDF]
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D. Mason, P. Andre, A. Bensussan, C. Buckley, C. Civin, E. Clark, M. de Haas, S. Goyert, M. Hadam, D. Hart, et al. CD antigens 2001 J. Leukoc. Biol., November 1, 2001; 70(5): 685 - 690. [Abstract] [Full Text] [PDF]
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 L. Marti, A. Abella, C. Carpene, M. Palacin, X. Testar, and A. Zorzano Combined Treatment With Benzylamine and Low Dosages of Vanadate Enhances Glucose Tolerance and Reduces Hyperglycemia in Streptozotocin-Induced Diabetic Rats Diabetes, September 1, 2001; 50(9): 2061 - 2068. [Abstract] [Full Text] [PDF]
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 N. Morin, J.-M. Lizcano, E. Fontana, L. Marti, F. Smih, P. Rouet, D. Prévot, A. Zorzano, M. Unzeta, and C. Carpéné Semicarbazide-Sensitive Amine Oxidase Substrates Stimulate Glucose Transport and Inhibit Lipolysis in Human Adipocytes J. Pharmacol. Exp. Ther., April 12, 2001; 297(2): 563 - 572. [Abstract] [Full Text]
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 P. Bono, K. Rubin, J. M. G. Higgins, and R. O. Hynes Layilin, a Novel Integral Membrane Protein, Is a Hyaluronan Receptor Mol. Biol. Cell, April 1, 2001; 12(4): 891 - 900. [Abstract] [Full Text]
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 N. Andrés, J. M. Lizcano, M. J. Rodríguez, M. Romera, M. Unzeta, and N. Mahy Tissue Activity and Cellular Localization of Human Semicarbazide-sensitive Amine Oxidase J. Histochem. Cytochem., February 1, 2001; 49(2): 209 - 218. [Abstract] [Full Text]
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P. Bono, S. Jalkanen, and M. Salmi Mouse Vascular Adhesion Protein 1 Is a Sialoglycoprotein with Enzymatic Activity and Is Induced in Diabetic Insulitis Am. J. Pathol., November 1, 1999; 155(5): 1613 - 1624. [Abstract] [Full Text] [PDF]
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 M. Moldes, B. Feve, and J. Pairault Molecular Cloning of a Major mRNA Species in Murine 3T3 Adipocyte Lineage. DIFFERENTIATION-DEPENDENT EXPRESSION, REGULATION, AND IDENTIFICATION AS SEMICARBAZIDE-SENSITIVE AMINE OXIDASE J. Biol. Chem., April 2, 1999; 274(14): 9515 - 9523. [Abstract] [Full Text] [PDF]
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P. Bono, M. Salmi, D. J. Smith, I. Leppanen, N. Horelli-Kuitunen, A. Palotie, and S. Jalkanen Isolation, Structural Characterization, and Chromosomal Mapping of the Mouse Vascular Adhesion Protein-1 Gene and Promoter J. Immunol., September 15, 1998; 161(6): 2953 - 2960. [Abstract] [Full Text] [PDF]
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