Structure, Diversity, and Repertoire of VH
Families in the Mexican Axolotl
Rachel Golub and Jacques Charlemagne
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 1998 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
J Immunol 1998; 160:1233-1239; ;
http://www.jimmunol.org/content/160/3/1233
Structure, Diversity, and Repertoire of VH Families in the
Mexican Axolotl1
Rachel Golub and Jacques Charlemagne2
T
he V region of the Ig heavy (H)3 chain of jawed vertebrates is generated by the random assembly of multiple V,
D, and J germline-encoded segments (1). This combinatorial diversity is greatly enhanced by such somatic events as the
deletion or addition of nucleotides at the V-D and D-J junctions,
point mutations, gene conversion, or editing. The germline repertoires of the VH segments have been studied, and the VH sequences
that have 75% or more identical amino acids in a given species are
assigned to the same VH family. This classification generally
agrees with classifications based on the similarities of the nucleic
acid sequences. These are determined using VH DNA probes on
genomic Southern blots with a washing stringency that allows
cross-hybridization between sequences that are $80%
homologous (2).
A limited number of representative vertebrate species have been
extensively analyzed. The number of VH families varies from one
species to another, and these variations do not seem to be related
to phylogeny. Two VH families have been described in the horned
shark (Heterodontus francisci) and in the skate (Raja erinacea)
and 1 in the nurse shark (Ginglymostoma cirratum); but 6 VH
families have recently been described in carcharine sharks, e.g.,
the sandbar shark (Carcharhinus plumbeus) and the bull shark
(Carcharhinus leucas) (3, 4). The best-studied teleost fish species
are the channel catfish (Ictalurus punctatus), with 7 VH families,
Comparative Immunology Group, National Centre for Scientific Research, Pierre and
Marie Curie University, Paris, France
Received for publication June 16, 1997. Accepted for publication October 14, 1997.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Université Pierre et Marie Curie and the Centre
National de la Recherche Scientifique (Unité de Recherche Associée 1134).
2
Address correspondence and reprint requests to Dr. Jacques Charlemagne, Immunologie Comparée, Université Pierre et Marie Curie and CNRS (URA 1135), boite 29,
9 quai Saint-Bernard, 75252 Paris Cedex 05, France.
3
Abbreviations used in this paper: H chain, heavy chain; FR, framework region;
CDR, complementarity-determining region; RACE, rapid amplification of cDNA
ends.
Copyright © 1998 by The American Association of Immunologists
and the rainbow trout (Oncorhynchus mykiss), with 11 VH families
(5, 6). The clawed toad, Xenopus laevis, has 11 VH families (7),
and the red-eared turtle (Pseudemys scripta) has at least 4 VH
families (8). A single functional VH gene has been found in the
chicken, which becomes diversified somatically by the conversion
of stretches of VH nucleotides provides by numerous nonfunctional VH pseudogenes (9). The best-known Igh locus in mammals
is the human one, which includes 64 VH segments (31 of which
being pseudogenes) that can be divided into 6 distinct families
(10). The laboratory mouse has 14 VH families, which is the most
diverse organization described so far in a vertebrate (11). Some
mammalian species, however, have a restricted number of VH families. The sheep VH repertoire is derived from a single germline
gene family with about 10 members, which is related to the human
VH4 family and the murine VHI subgroup (12). All of the approximately 100 VH genes of the rabbit seem to belong to the same
family, homologous to the human VH3, but the 39-most VH1 gene
is used preferentially and somatically diversified by gene conversion (13). There is also a single VH family in the pig, and it is
related to the human VH3 family, the rabbit VH1 gene, and the
single functional gene of the chicken (14).
Studies on Xenopus and on the Mexican axolotl (Ambystoma
mexicanum) have demonstrated that both species synthesize class
IgM and IgY Ig, but that a third class, IgX, is produced only by
Xenopus. The amino acid sequences of the axolotl Cm and Cy
chains are remarkably similar to those of their respective Xenopus
counterparts (15, 16). The Igh locus of Xenopus includes a large
number of VH segments, divided into 11 families of about 1 to 40
members (7, 17) and, although subjected to some limitation (18),
the Xenopus-specific Ab repertoire approaches the avian and mammalian schemes in its complexity (17). Like the more advanced
vertebrates, Xenopus synthesizes IgM class Abs during the primary
response and IgG-like IgY Abs in the secondary response to thymus-dependent Ags (19). This is not so in the axolotl, where almost all specific Abs belong the IgM class (20). No typical Ab
enhancement occurs after antigenic challenge, IgY Abs are not
involved, and responses do not depend on thymus-derived cells,
0022-1767/98/$02.00
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The Mexican axolotl VH segments associated with the Igh Cm and Cy isotypes were isolated from anchored PCR libraries
prepared from spleen cell cDNA. The eight new VH segments found bring the number of VH families in the axolotl to 11. Each
VH had the canonical structural features of vertebrate VH segments, including residues important for the correct folding of the
Ig domain. The distribution of ser AGC/T (AGY) and TCN codons in axolotl VH genes was biased toward AGY in complementarity-determining region-1 (CDR1) and TCN in framework region-1 (FR1); there were no ser residues in the FR2 region. Thus,
the axolotl CDR1 region is enriched in DNA sequences forming potential hypermutation hot spots and is flanked by DNA
sequences more resistant to point mutation. There was no significant bias toward AGY in CDR2. Southern blotting using familyspecific VH probes showed restriction fragments from 1 (VH9) to 11–19 (VH2), and the total number of VH genes was 44 to 70,
depending on the restriction endonuclease used. The VH segments were not randomly used by the Hm and Hy chains; VH1, VH6,
and VH11 were underutilized; and the majority of the VH segments belonged to the VH7, VH8, and VH9 families. Most of the nine
JH segments seemed to be randomly used, except JH6 and JH9, which were found only once in 79 clones. The Journal of
Immunology, 1998, 160: 1233–1239.
1234
VH REPERTOIRE IN THE MEXICAN AXOLOTL
even those to potentially thymus-dependent Ags (21–23). Furthermore, the very simple, constant isoelectric focusing (IEF) patterns
of the H and L chain spectrotypes from anti-DNP Abs suggest that
specific Abs use a restricted repertoire of VH and VL elements
(24). Thus, although anuran and urodele amphibians are considered monophyletic and as having diverged from primitive
salamander-like ancestors some 270 million years ago (25), the
potential of their respective VH repertoires seem to have strongly
diverged during evolution. We have recently described the cDNA
transcripts of 3 VH families in the axolotl (26). The present work
describes the VH repertoire of this urodele species using VH cDNA
libraries obtained by anchored PCR and also examines the
genomic diversity using Southern hybridization.
solution (0.4 M NaOH). The membrane was briefly washed in 23 SSC and
UV cross-linked. The membrane was incubated for 4 to 6 h at 65°C in a
prehybridization solution (1.53 SSC, 13 SDS, 103 dextran sulfate, 0.5%
tetrasodium pyrophosphate). DNA-homologous probes were prepared by
PCR using 59 primers specific for the CDR1 regions and 39 primers complementary to the FR3 regions of each VH family (data not shown). Fifty
nanograms of DNA were labeled with a[32P]ATP by random hexanucleotide priming to 108 cpm/mg. Hybridization was performed for 16 to 24 h
at 65°C in hybridization solution (1.53 SSC, 13 SDS, 103 dextran sulfate, 0.5% tetrasodium pyrophosphate, 0.5% blotto, and 5 mg denatured
sonicated Escherichia coli DNA). Membranes were then washed in 43
SSC, 0.05% SDS at 65°C for 15 min, and in 23 SSC, 0.025% SDS at 65°C
for 15 min, and then autoradiographed for 2 to 6 days at 280°C.
Materials and Methods
A total of 19 independent consensus L-VH nucleotide sequences
were obtained by analyzing 38 cDNA clones selected from the
VHm library and 41 clones selected from the VHy library (all of
these clones had different sequences). The amino acid sequences
deduced from these clones are shown in Figure 1, together with 3
of the previously described VH sequences, now numbered VH1
(VA1.3), VH2 (VB1.1), and VH3 (VC3.1) (26). Sequences are
numbered chronologically and aligned to VH1 in order of decreasing similarity. Among the 19 new VH sequences, 13 were no more
than 75% homologous to the previously characterized VH1, VH2,
and VH3 families and gave rise to 8 new families. Sequences
within any given axolotl VH family all had at least 80% identical
amino acids, except for clone VH2 (73Y), which was only 77.2%
homologous to VH2 (29M), but clearly belonged to the VH2 family. There were 3 different sequences in the VH2 family, 3 in the
VH3, 2 in the VH4, 2 in the VH8, and 4 in the VH9 families. Most
families were less than 45% homologous, except for VH3 and VH5
(60 – 64% homologous), VH1 and VH8 (about 53%), and VH5 and
VH7 (53.7%). Conversely, some families were more dissimilar,
such as VH8 and VH9 (24.7–27.1%) or VH10 and VH11 (25.5%).
A clone from the VHy cDNA library (F.24Y) gave a deduced
amino acid sequence that was easily aligned to other VH sequences, but positions 22 (Ser instead of Cys) and 92 (Gly instead
of Cys) were not conserved, and a stop codon appeared at position
69. This sequence is thus from a nonfunctional VH pseudogene and
was not attributed to a defined family. The axolotl VH segments of
all 11 families all contained the canonical features of vertebrate
Construction of VH libraries
Total cytoplasmic RNA from the spleen cells of 10-mo-old unimmunized
axolotls was used to built two VH libraries by anchored PCR using a commercial kit (59 system for rapid amplification of cDNA ends (RACE), Life
Technologies, Cergy-Pontoise, France), essentially following the manufacturer’s instructions with minor modifications (27). Briefly, first-strand
cDNA was synthesized using specific downstream primers complementary
to the axolotl Cm1 or Cy1 regions (CM1, 59-GACTCGAGTCGACGTTC
CCAAGGA-AGGGTGTGAGAC; CY1, 59-CAGAGGTTCTTGGGCAC)
and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Superscript II; Life Technologies). The RNA template was removed, and the
first-strand cDNA was purified. The second-strand cDNA was then synthesized using Taq DNA polymerase Amplitaq Roche, Perkin Elmer,
Courtaboeuf, France and the manufacturer’s upstream 59 RACE anchor
primer. The double-stranded cDNA was then amplified using the manufacturer’s UAP primer and the 39 CM1 or CY1 primers. The amplified VH
segments were cloned in the NotI and SalI sites of the pBluescript KS2
vector (Stratagene, Basel, Switzerland) and sequenced.
Southern blotting
Genomic DNA was isolated from erythrocytes obtained from a single axolotl. Cells were lysed in a 1% SDS lysis buffer (50 mM Tris, pH 7.5, 100
mM EDTA) for 45 min at 65°C and digested with proteinase K (100 mg/
ml) for 24 h at 45°C. DNA was isolated as described (28). The DNA (20
mg) was digested overnight with 320 U of restriction enzymes, BamHI,
BglII, EcoRI, and EcoRV, and electrophoresed in a 0.8% agarose gel in
TAE buffer (40 mM Tris pH 8.0, 1 mM EDTA). DNA was depurinated by
soaking the gel in 0.25 M HCl for 10 min and then denatured in 0.4 M
NaOH for 15 min. DNA was transferred onto Zeta Probe membranes (BioRad Iury sur Seine, France) by DNA capillary transfer for 6 h in alkaline
Results
Definition of 11 axolotl VH families
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FIGURE 1. Multiple alignment of the amino acid sequence of the axolotl L-VH segments. Residues identical to the VH1 (clone 182 M) sequence are
denoted by hyphens, and spaces introduced to optimize similarity between sequences are indicated by dots. FR/CDR regions (30) and the number of the
most conserved residues are indicated above the sequence. Under the sequences, residues conserved in most sequences are indicated by 5. GenBank
accession numbers are: AF027252 (VH1.182 M), AF027253 (VH2.29 M), AF027254 (VH2.73Y), AF027255 (VH3.49 M), AF027256 (VH3.73 M),
AF027257 (VH4.33 M), AF027258 (VH5.8Y), AF027259 (VH5.43 M), AF027260 (VH6.65Y), AF027261 (VH7.15 M), AF027262 (VH8.46 M), AF027263
(VH8.40 M), AF027264 (VH9.85 M), AF027265 (VH9.42 M), AF027266 (VH9.1 M), AF027267 (VH9.103 M), AF027268 (VH10.33Y), AF027269 (VH11.5
M), AF027270 (VH9.38 M), AF027271 (VHF.24Y).
The Journal of Immunology
1235
VH, including Cys22 (FR1), Cys92 (FR3), Trp36 (FR2), and Trp47
(FR2) (Fig. 1). Several structurally important residues were frequently present, like Gln1, Pro14, Gly44, Arg66, Tyr/Phe90, Tyr91,
Ala93, and Arg94. The last five residues form the YYCAR stretch,
which is present in most vertebrate VH sequences. Some uncommon residues were present. VH1, VH4, VH6, and VH10 has a Ser
(or Thr) residue at position 26 (FR1) instead of the Gly, which is
always found at this position in mammals. Extra Trp residues were
found at positions 10 (FR1) of VH4, 14 (FR1) of VH10, 38 (FR2)
of VH8, 42 (FR2) of VH11, and 52 (CDR2) of VH4. Extra Cys
residues were found at position 5 (FR1) of VH10, in the CDR1
region of VH8 (3 consecutive Cys), at position 52 (CDR2) of VH8
and VH11, and position 67 (FR3) of VH4. Cys residues are also
present in the CDR1 of Xenopus VH3 and the rainbow trout VH3
and VH10 segments; at position 52 (CDR2) of rainbow trout VH3
and VH10, and at position 67 of the Xenopus VH9 segment (6, 7).
Finally, the Pro residues found in the CDR1 region of the axolotl
VH6 and VH7 segments have no equivalent in other
vertebrate CDR1.
used. VH1, VH6, and VH11 were clearly underutilized (only one
VH6 and one VH11 segment were found among the 79 clones
analyzed), and VH7, VH8, and VH9 were overused. The VH4 and
VH5 segments seem to be underutilized by the m chains. Most of
the 9 JH segments (Ref. 29 and R. Golub, unpublished data)
seemed to be randomly used, except for JH1 and JH5, which were
overused, and JH6 and JH9, which were used in only 1 of the 71
clones bearing identifiable JH (Table II). There was no clear preferences in the VH/JH combinations, except that 6 of the 8 VH5/Cy
clones all had JH1, but had different VDJ junctions (the 2 other
VH5/Cy clones had undetermined JH segments; data not shown).
There was little redundancy among the clones. However, the VHy
library contained 14 clones bearing the VH2/JH5 combination, with
identical VDJ junctions.
Table I. Complexity of the axolotl VH families
Genomic analysis of the VH segment repertoire
DNA sequences representative of the 11 axolotl VH families were
used to probe restriction endonuclease-digested DNA obtained
from a single animal, and interpretable Southern blots hybridization patterns were obtained for each of the probes. Most of these
probes revealed multiple hybridizing fragments, and there appeared to be little similarity between the hybridization patterns
revealed by the various probes (Fig. 2). The number of restriction
fragments ranged from 1 for VH3 and VH9 to 11 to 19 for VH2
(Table I). The total number of VH genes, estimated by adding the
number of hybridizing fragments from each family, was 44 to 70,
depending on the restriction endonuclease used. However, this
number may be underestimated for several reasons (see
Discussion).
VHm and VHy repertoires
The VH and JH segments usage of the 38 VHm and 41 VHy clones
is shown in Table II. The different VH segments were not randomly
Complexitya
Probes
VH
Family
VH1
VH2
VH3
VH4
VH5
VH6
VH7
VH8
VH9
VH10
VH11
Clone
VH1.3
73Y
49M
33M
43M
65Y
15M
46M
103Md
33Y
5M
Size (nt)
282
288
266
288
270
273
279
273
204
275
273
HI
BII
b
5
11b
—
3b
5
1
11
6
1
4
3
c
—
12
1
3
—
2b
—
11
1
—
—
RI
RV
b
6
10
1
3
6
3
9
8
1
3b
3
6
19
2
3
6
—
6
—
1
2
4
a
Estimated number of VH genes within each family is based on the number of
hybridizing BamHI (HI), BglII (BII), EcoRI (RI), and EcoRV (RV) fragments detected by Southern blot (Fig. 2), corrected by the visual examination of the radioautographs at different exposure times.
b
An enzyme cutting site is present in the corresponding VH segment.
c
—, not done.
d
Clone 103M is identical to clone VH9 (85M).
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FIGURE 2. Southern blot analysis of axolotl VH gene families. Erythrocyte DNA from a single axolotl was digested to completion with BamHI, (HI),
BglII (BII), EcoRI (RI), and EcoRV (RV), fractionated in agarose gels, and transferred onto nylon membranes. Membranes were probed with DNA
representing 11 VH families.
1236
VH REPERTOIRE IN THE MEXICAN AXOLOTL
Table II. VH family usage in the VHm and VHy libraries
VH
1
(5–6)a
2
(11–19)
3
(1–2)
4
(3)
5
(5–6)
6
(1–3)
7
(6–11)
8
(6–11)
9
(1)
10
(2–4)
11
(3–4)
F
(ND)
nb
m
2
J4d
J5
3
J1
J4
J5
5
J1
J3
J7
J8(2)e
1
J5
1
J3
0
12
J1(2)
J2(3)
J4
J5(4)
J7
J8
6
J2
J3
J5(3)
J8
6
J1(4)
J5
J7
0
1
J2
1
J9
38
(38)
y
0
4
J5(4)
2
J5
J8
7
J1
J4(3)
J6
J8
8
J1(6)
1
J1
5
J2
J3
J4(2)
5
J2(2)
J4
4
J1
J2
J5
3
J1
J4
0
2
J3(2)
41
(33)
c
a
Predicted numbers of VH segments (see Table I).
n indicates the total numbers of clones analyzed from each library (the number of clones bearing recognizable JH segments are indicated in parentheses).
Number of clones expressing a given VH family.
d
JH segments associated with the VH segments.
e
Number of clones with identical JH segments.
b
c
Most of the mouse and human CDR1 regions, as defined by Kabat
et al. (30), are 5 amino acids long, excepted for some VH families
(human VH2 and VH6, mouse VH3, VH8, and VH12), which may
have up to seven residues. The axolotl CDR1 regions, as defined
following the same criteria, varied from three (VH6) to nine (VH2
and VH4) residues. Ag-interactive sites in VH segments can also be
assessed by analysis of Ig structures determined by x-ray crystallography. CDR1 can be treated as a part of a single region covering
residues 26 to 35; the part of this region that forms a hairpin loop
outside of the framework b-sheet spans residues 26 to 32 (region
H1). In the same way, residues 52 to 56 of the CDR2 region (50 –
65) form hairpin loop H2 (31). These H1 and H2 hypervariable
regions form a small repertoire of conformational canonical structures in mammals (32), and it was recently shown that the VH
segments of cartilaginous fish have H1 and H2 sequences that fit
well with those commonly found in the human and mouse (3). A
systematic comparison of the presumed axolotl H1 and H2 sequences with the mammalian H1 and H2 sequences of the different
canonical classes showed that the axolotl VH1 segment had a
three-residue H2 region (Tyr53-Ser-Gly) that is identical to the
three-residue hairpin that formed the H2 loop of the HyHEL-10
molecule (32) and is associated, as in HyHEL-10, to an arginine
residue at position 71. The axolotl VH7 H1 loop presents a stretch
of seven residues at position 26 to 32 (Gly26-Phe-Ser-Phe-GluAsp-Tyr) that is very similar to the H1 regions of the KOL and
NEWM molecule (33) and may adopt an equivalent conformation,
considering that residue Met34 and Arg94 are also conserved. For
the other VH families, the presumed H1 and H2 regions present no
significant similarities with the mammalian canonical structures,
although some isolated residues were well conserved (data not
shown).
It has been suggested that the IgV genes have evolved so that
their DNA sequence favors somatic mutation in those parts of the
V segments in which the mutations are likely to affect Ag binding
(34). Thus, the choice of triplets encoding the ser residue is significantly biased in the VH and Vk CDRs of several species, because AGC/T (AGY) instead of TCN falls within the intrinsic
hypermutation hot spot consensus (Pu-G-Py-A/T) (35). The distributions of AGY and TCN triplets in axolotl VH genes showed a
clear preference for AGY in CDR1/H1 (TCN/AGY 5 0.73), a
significant bias for TCN in FR1 (TCN/AGY 5 8.25), a slight bias
for TCN in CDR2/H2 (TCN/AGY 5 2.71), and no significant bias
in FR3 (TCN/AGY 5 1.66). There was no Ser residue in the
axolotl FR2 regions (Fig. 3).
Phylogenic analysis
The amino acid sequences for the 11 axolotl VH families were
compared with the GenBank database using the FASTA alignment
program (36). Except for VH6, VH10, and VH11, no axolotl VH
had a vertebrate VH homologue that had more than 55% identical
amino acids. The most striking similarities were between axolotl
VH1, VH2, and VH8 and the human VH4 family and between axolotl VH1 and the single sheep VH family, which is homologous to
the human VH4 family (12). However, there were also significant
similarities with lower vertebrate VH, such as the rainbow trout
VH5 and VH8, Xenopus VH1 and VH3, and the coelacanth and
FIGURE 3. Distribution of ser residues in the 11 aligned axolotl VH families. FR regions and the number of residues limiting the FR and CDR/H regions
are indicated above the sequences. Non-ser residues are indicated by hyphens; s and S indicate Ser residues encoded by TCN and AGY, respectively. The
limits of the CDR (30) and H (33) regions and the s/S ratio are indicated under the sequences.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Analysis of the CDR/H regions
The Journal of Immunology
1237
horned shark VH. Axolotl VH are aligned with their closest homologues in Figure 4. FR1 and FR2 were the most conserved regions:
for example, the FR1-CDR1-FR2 regions of axolotl VH1 and human VH4 were 79.1% homologous. The 14-amino acid-long FR2
regions were also very similar in some pairs: they were identical in
the axolotl VH10 and in a VH segment belonging to the mouse VH4
family (data not shown), and there was a single amino acid difference between the FR2 of the axolotl VH1 and the human VH4
segments. The FR3 regions were also well conserved in some
cases; the axolotl VH3 and trout VH8 segments were 80%
homologous.
Discussion
The 79 cDNA clones isolated from the IgHm and IgHy libraries
indicate that the axolotl has a relatively large number of VH segments, with 11 families and a pseudogene. All of the families seem
to associate nonselectively with Cm or Cy, except for VH6, VH10,
and VH11, which are infrequently expressed. The most commonly
expressed VH families are VH7, VH8, and VH9. There was some
evidence of nonuniform usage of VH families, but the sample was
too small to determine the preferential association of some families
to one or the other isotype (37). The same panel of JH segments
was used in both isotypes, except for JH6, which was found in only
2 IgHy clones.
It is clear from that the axolotl m and y chains use the same
collection of VH and JH segments and may have an Igh locus
organized along the same lines as in mammals. However, this
raises the problem of isotype switching in axolotl, where, in contrast to Xenopus, IgY are not used in the secondary responses (20),
are insensitive to thymectomy (38), and can be produced by an
independent population of B cells in the developing animals (38).
Abundant IgY molecules are found in the stomach and intestinal
epithelia of young axolotls, and they can be secreted into the gut
lumen in association with secretory component-like molecules
(39). These IgA-like secretory IgY molecules appear in the blood
late in development (7 mo), and the amounts of IgM and IgY in the
blood are similar after the animals reach 10 mo. Our results indicate that the VH usage does not seem to be restricted to the y or m
isotype and that IgY Abs may provide a natural repertoire of wide
specificity in unimmunized animals. Interpretable Southern blots
were obtained for each of the VH family defined from the cDNA
analysis, despite the technical difficulty caused by the large size of
the axolotl genome (40). Relatively few members were detected in
each family, but the 44 to 70 potential bands detected in the Southern blots may be an underestimate of the VH repertoire for several
reasons: rare VH families may not have been detected in the cDNA
libraries and thus not probed in the genome, and the presence of
several hybridizing VH segments in some of the labeled bands
cannot be excluded. However, the repertoire could also have been
overestimated because outbred axolotls were used, so that a single
VH may give rise to different bands, and also because there are
restriction endonuclease sites in some of the VH segments (although Table I shows that this had a little effect on the bands
counted). A single hybridizing band was found for VH9 with all of
the restriction enzyme used, although four cDNA sequence variants were found. Thus, the VH9 family could be represented by a
single polymorphic member.
Some of the less frequently used axolotl VH segments (VH6,
VH10, VH11) have species-specific amino acid sequences that are
somewhat different from all the VH in the GenBank database. Conversely, VH1, VH2, and VH8 are 55 to 65% similar to the human
VH4 family, the single sheep VH4-like family, and also to other
vertebrate VH such as the Xenopus VH4 and the mouse VH2 (Q52)
families (Fig. 4). The similarity between the nucleotide sequences
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FIGURE 4. Alignment of VH sequences from different vertebrate species presenting the best FASTA scores (36) with axolotl VH sequences belonging
to different families. For each pair of sequences, residues identical to the axolotl sequence are indicated by hyphens, and spaces introduced to optimize
homology between the aligned sequences are denoted by dots. Percentages of similarity between sequences are indicated on the right. Synonymous
substitutions are indicated below the sequences by asterisks.
1238
nodes, Peyer’s patches, and germinal centers (50). These mice
show specific IgM responses equal to or greater than those of wildtype mice, but have impaired high affinity IgG1 production (50),
and their immune response has interesting similarities to those of
normal cold-blooded vertebrates. However, the defect of LTa2/2
mice can be partially corrected by hyperimmunization with large
doses of Ag. These mice show somatic mutations typical of affinity
maturation, although the mutations are less numerous than those
found in wild-type mice (51). Thus, point mutation and affinity
maturation are not absolutely dependent on germinal centers in
mice, and this might be also the case in cold-blooded vertebrates
such as fish and amphibians, which naturally lack primary lymphoid follicles and Ag-driven germinal centers.
Acknowledgments
We thank Louis Du Pasquier (Basel Institute for Immunology) and Bénédicte Sammut (Université de Bourgogne) for useful discussion and help in
performing the Southern blots, Julien Sadreddine Fellah for advice and
help, and Brigitte Cuvelier and Jean Desrosiers for editing assistance.
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of axolotl VH1 and human VH4 (41) reaches 80% identity, which
defines a VH family in a given species. The FR1 to FR2 amino acid
sequences of these same VH segments are 79.1% homologous, but
the FR3 regions are very different, although most of the amino acid
residues are conservative replacements (Fig. 4). This great similarity between sequences of VH families from species so phylogenetically different may have physiologic significance. The human
VH4 family contains about 12 members (10) and displays little
polymorphism. It is preferentially used by CD51 (B1) pre-B cells
(42), frequently used by B ALL and B CLL lymphomas (43), and
strongly associated with anti-DNA and anti-red cell (anti-I/i cold
agglutinins) autoantibodies (44, 45). The serum of unimmunized
axolotls contains significant amounts of natural Abs against bacterial and erythrocyte Ags, but also against DNA and horseradish
peroxidase (J. Charlemagne and A. Tournefier, unpublished data).
The profile of the VH4 family in the serum of unimmunized axolotl
should now be monitored and their participation in natural Abs
analyzed.
The VH1, VH6, VH8, and to a lesser extent, VH2 axolotl FR1
regions have amino acid sequences that are similar to the consensus FR1 sequence that defines the human and murine clan II (46).
Clan II-like VH sequences seem to emerge early in phylogeny
(Ref. 47, and T. Roman et al., manuscript in preparation); thus, a
strong selective pressure seems to have operated throughout much
of vertebrate evolution to preserve the structure of VH segments
that may play a key role in species survival. Figure 4 shows that
the 14-amino acid-residue FR2 sequence is present almost intact in
VH segments in phylogenetically very distant species such as the
rainbow trout, coelacanth, Xenopus, human, and mouse, regardless
of their family or clan membership. A stretch of 4 to 7 residues
from FR2 is involved in VH-VL contacts in mammals and is thus
important for the three-dimensional structure of Ig molecules (48).
This indicates that the three-dimensional configuration of the
VH-VL contact, which is important for building the Ag-combining
site, is conserved in most vertebrate Ig.
Although some of the canonical structures of the H1 and H2
hypervariable regions in sharks and mammals are similar (3), this
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regions varies more than in mammals. This could mean that the
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