Download in the Cartilaginous Fish Forms Transmembrane and Secretory

Unprecedented Multiplicity of Ig
Transmembrane and Secretory mRNA Forms
in the Cartilaginous Fish
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of June 18, 2017.
Lynn L. Rumfelt, Marilyn Diaz, Rebecca L. Lohr, Evonne
Mochon and Martin F. Flajnik
J Immunol 2004; 173:1129-1139; ;
doi: 10.4049/jimmunol.173.2.1129
http://www.jimmunol.org/content/173/2/1129
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References
The Journal of Immunology
Unprecedented Multiplicity of Ig Transmembrane and
Secretory mRNA Forms in the Cartilaginous Fish1
Lynn L. Rumfelt,2*† Marilyn Diaz,3† Rebecca L. Lohr,† Evonne Mochon,† and
Martin F. Flajnik†
I
n the animal kingdom, only the jawed vertebrates have an
adaptive immune system grounded upon somatically generated Ag receptors (1, 2). All jawed vertebrates also have multiple Ig H chain isotypes (1). Whereas IgM is present in all jawed
vertebrates, with one exception (a form of IgD in teleosts, Ref. 3),
the other mammalian isotypes are not found in ectotherms, with
amphibians having IgX and IgY, and cartilaginous fish having two
distinct isotypes, IgW and IgNAR (1, 2). Based on phylogenetic
analysis, IgW and IgNAR previously were believed to be present
only in cartilaginous fish (4); however, recently IgW was isolated
from lungfish (5), showing that both IgW and IgM ancestral genes
were present in the common ancestor of all extant jawed vertebrates. The IgW H chain long form is composed of seven domains
in all cartilaginous fish so far studied (6 – 8) and of eight domains
in the lungfish (5). An IgWshort form having three domains also has
been described at the cDNA and genomic levels in skates (previously called IgX), presumably a splice variant of the long form (8,
9); in addition, a protein identified as a second Ig isotype besides
IgM in batoids (rays and skates) and frill sharks is postulated to be
this short form of IgW (10, 11). The long form of the IgW protein
*Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33101; and †Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Received for publication January 28, 2004. Accepted for publication May 14, 2004.
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
M.D. was a postdoctoral fellow supported by Burroughs Wellcome Fund Fellow of
the Life Sciences Research Foundation. M.F.F. was supported by National Institutes
of Health Grant RR06603.
2
Address correspondence and reprint requests to Dr. Lynn L. Rumfelt at the current
address: Department of Immunology, University of Toronto, Sunnybrook and Women’s Health Sciences Centre, 2075 Bayview Avenue, Room A331, Toronto, Ontario
M4N 3M5 Canada. E-mail address: [email protected]
3
Current address: Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 27709-2233.
Copyright © 2004 by The American Association of Immunologists, Inc.
has also been isolated from metabolically labeled nurse shark
splenocytes (6). To date, IgNAR has been described only in elasmobranchs (sharks, skates, and rays), and it is an unusual Ig composed of an H chain dimer that does not associate with L chains
(12). The IgNAR V recognizes Ag as a single domain (see Fig. 8)
and its gene mutates to very high levels, suggesting that it is important for the shark adaptive immune system (12–14).
It has been known for many years that the B cell Ag receptor
exists both in a transmembrane (Tm)4 form that acts to activate/
tolerize lymphocytes and as a secretory (Sec) form that binds to Ag
and is responsible for the well-known effector functions of the
humoral immune system (15). The Tm and Sec forms of all Ig
isotypes studied to date are derived from mRNAs that are alternatively spliced. In almost all species, the membrane-proximal
constant (CH) domain is contiguous with a Sec segment, and this
CH domain and Sec piece are invariably encoded within one exon
(16). Upstream of the nucleotides encoding the Sec tail is an “alternative” or cryptic splice site, which is used to splice the C exon
onto Tm exons. In the teleost fish, instead of splicing the Tm exons
into the ultimate C domain exon, Tm exons are rather spliced onto
the penultimate (CH3) exon (3). The function of this form is not
known, but it is a universal feature of IgM Tm mRNA forms in this
vertebrate class. In other bony fish more primitive than the teleosts
(i.e., the holosteans), there can be multiple spliced forms of the
IgM Tm mRNA (17).
Although cartilaginous fish ␮-chain mRNA is spliced in the typical (human/mouse) fashion, as described, previous studies showed
that skate IgW H chain cDNA had two secretory forms, one with
two predicted CH domains and another with six (8). Recent lungfish work demonstrated two IgW Sec cDNA forms as well (5),
although the short cDNA form had not been found previously in
4
Abbreviations used in this paper: Tm, transmembrane; Sec, secretory; FR, framework region of V domain; NC, noncanonical; ␻VH, IgW H chain variable domain;
␯VH, IgNAR H chain variable domain; CART, conserved Ag receptor motif; Igsf, Ig
superfamily; UT, untranslated region.
0022-1767/04/$02.00
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In most jawed vertebrates including cartilaginous fish, membrane-bound IgM is expressed as a five Ig superfamily (Igsf)-domain
H chain attached to a transmembrane (Tm) region. Heretofore, bony fish IgM was the one exception with IgM mRNA spliced to
produce a four-domain Tm H chain. We now demonstrate that the Tm and secretory (Sec) mRNAs of the novel cartilaginous fish
Ig isotypes, IgW and IgNAR, are present in multiple forms, most likely generated by alternative splicing. In the nurse shark,
Ginglymostoma cirratum, and horn shark, Heterodontus francisci, alternative splicing of Tm exons to the second or the fourth
constant (CH) exons produces two distinct IgW Tm cDNAs. Although the seven-domain IgW Sec cDNA form contains a canonical
secretory tail shared with IgM, IgNAR, and IgA, we report a three-domain cDNA form of shark IgW (IgWshort) having an unusual
Sec tail, which is orthologous to skate IgXshort cDNA. The IgW and IgWshort Sec transcripts are restricted in their tissue distribution and expression levels vary among individual sharks, with all forms expressed early in ontogeny. IgNAR mRNA is alternatively spliced to produce a truncated four-domain Tm cDNA and a second Tm cDNA is expressed identical in Igsf domains as
the Sec form. PBL is enriched in the Tm cDNA of these Igs. These molecular data suggest that cartilaginous fish have augmented
their humoral immune repertoire by diversifying the sizes of their Ig isotypes. Furthermore, these Tm cDNAs are prototypical and
the truncated variants may translate as more stable protein at the cell surface. The Journal of Immunology, 2004, 173: 1129 –1139.
1130
DIVERSITY OF CARTILAGINOUS FISH Ig GENE RNA PROCESSING
sharks until this report. The Tm cDNA forms of IgW have not been
described in any species, but we assumed that they would be
spliced to the ultimate CH domain, like the IgM (18) and IgNAR
(14, 19) Tm cDNA previously identified. In this paper we re-examined this problem and found that, like teleost IgM, there are
multiple-spliced Tm cDNA forms of IgW and IgNAR, possibly
selected over evolutionary time to limit proteolysis of the putative
cell surface proteins. In addition, our findings reveal that each
elasmobranch group, as well as the lungfish, expresses the long
and short Sec IgW mRNA forms, and their expression levels in the
nurse shark varies among individuals with no apparent pattern.
Materials and Methods
Animals
Nurse shark pups (Ginglymostoma cirratum) were delivered by Caesarean
section from a female shark as described (20). The age of pups, feeding and
caretaking were performed as described (21). Adult nurse sharks were captured off Little Torch Key, FL and horn sharks were obtained from Pacific
Biomarine (Monterey, CA). The animals were sacrificed with an overdose
of tricane methyl sulfonate (MS-222) anesthesia (20).
Tissues were dissected from adult nurse and horn sharks, and from nurse
shark pups, and total RNA was isolated as described (21). The newborn
pup spleen and epigonal cDNA libraries, and adult horn shark (Heterodontus francisci) and nurse spleen and PBL cDNA libraries were constructed
as described previously (12, 21–24). Probes for library screens and Figs. 6
and 7 are listed in Table I and were labeled with 32P[dCTP] as described
(21). Spleen, epigonal organ, and PBL oligo(dT)-primed cDNA was made
from 5 ␮g of mRNA as described and used as templates for PCR amplification (21). Primers for RT-PCR are shown in Table II. Expected sizes for
PCR products are listed in Table III. Positive controls to verify correct PCR
amplification conditions were: IgW Sec clone 14S, IgW Tm clone 1E
(GenBank accession no. AY524297), IgNAR Sec clone 3-4 (GenBank accession no. U18701; Ref. 12), and full-length IgNAR Tm clone 7A (GenBank accession no. U18721; Ref. 12). Nurse shark total RNA was used (20
␮g/lane) for Northern analyses. The labeled probes were hybridized with
membranes for 20 h minimum at 42°C, then washed under high stringency
conditions (25). Nurse shark NDPK RNA loading control was a gift from
M. Kasahara (Graduate University of Advanced Studies (Sokendai),
Hayama, Japan) (26). To identify horn shark and nurse shark Tm IgW and
IgWshort, horn shark and nurse shark adult spleen cDNA libraries were
plated and lifted as described using two membranes per plate (22) and
hybridized to IgW VH and CH6 probes separately. Isolated clones were
selected based on ␻VH⫹ and ␻CH6⫺ thereby enriching for the short forms.
Alignments and phylogenetic trees
IgW Sec tail cDNA sequences from library screenings were aligned with
Sec tails from sequences with the following Swiss-Prot and GenBank accession numbers: Hf IgWsh1 P83742; Hf IgWsh2 P83743; Hf IgWsh3
P83744; Hf IgM X0778; Gc IgWshort clone 2-4 AY524287; Gc IgWshort
clone 1-2 AY524282; Gc IgWshort clone 1-3 AY524289; Gc IgWshort pnc
P83984; Gc IgWshort spl P83985; little skate, Raja erinacea, IgXshort clone
20 AAA49546; nurse shark IgW U51450; nurse shark IgM M92851; sandbar shark, Carcharhinus plumbeus, IgW 1117935; little skate IgM
M29677; horn shark IgM X0778; ratfish, Hydrolagus colliei, IgM
Results
cDNA analysis reveals that IgW Tm and one form of IgNAR Tm
are smaller than their secreted forms, and shark IgWshort is
orthologous to skate IgWshort
While screening neonatal nurse shark spleen and epigonal organ
cDNA libraries with an IgW VH probe to examine the early VH
gene repertoire, we isolated IgW Tm cDNA clones. In retrospect,
this was not unexpected because these mRNA sources from young
animals are highly enriched for IgM Tm mRNAs (21). The deduced proteins encoded by most of the cDNA clones unexpectedly
were found to have the Tm region contiguous with the CH4 rather
than the CH6 domain, the C-terminal domain in IgW Sec cDNA
forms (see Fig. 8). Subsequently, additional cDNA clones were
isolated from the adult nurse shark PBL cDNA library screened
with an IgW VH probe and from adult nurse shark and horn shark
spleen cDNA libraries screened differentially with IgW VH and
CH6 probes (positive for the former and negative for the latter),
thereby selecting for the smaller sizes of IgW cDNA. These
screenings resulted in identification of more nurse shark IgW Tm
(five domains) and Sec (seven domains) cDNA clones; unpredictably, three-domain Tm and Sec cDNA clones also were found that
we designate IgWshort Tm and Sec (Fig. 1, see Fig. 8). These short
Sec and Tm cDNA forms were unanticipated in sharks (4), and
previously were believed to be present only in the skate as Sec
cDNA (2, 4). The models we present here for the different sized Ig
obviously H chain Tms derived from the cDNA data are tentative
Table I. Probesa
Name
NS
NS
NS
NS
HS
NS
NS
NS
NS
a
IgW Tm
IgM VH
IgNAR VH
IgW VH
IgM VH
IgM TM
NDPK
IgW CH6
IgNAR Tm
Forward and Reverse Primers
Template
␻TM1E For ⫹ ␻3⬘ UT TM1E Rev
␮VH FR1 For ⫹ ␮VH FR4 (50S) Rev
␯FR1VH (3⬘-4) For ⫹ ␯FR3VH (3-4) Rev
␻NEW12A FR1 For ⫹ ␻12A CH1 Rev
␻HS VH For ⫹ ␻HS VH Rev
␮TM For (35S) ⫹ ␮3⬘ UT TM Rev (35S)
NS NDPK For ⫹ NS NDPK Rev
␻CH6 For (38E) ⫹ ␻CH6 Rev (38E)
␯TM (7A) For ⫹ ␯3⬘ UT TM (7A) Rev
1E (NS pup epig org lib)
50S (NS pup spln lib)
3– 4 (Accession no. U18701)
12A (NS spln lib)
3–5A (HS spln lib)
35S (NS pup spln lib)
Accession no. M63964
38E (NS pup epig org lib)
7A (U18721)
For, forward; Rev, reverse.
Anneal
63°C
55°C
64°C
52°C
62°C
53°C
64°C
57°C
58°C
30
30
30
70
30
30
30
30
30
s
s
s
s
s
s
s
s
s
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cDNA library screens, Northern blot analysis, and RT-PCR
AAC12920; guitarfish, Rhinobatus productus IgNAR AY524298; Siberian
sturgeon, Acipenser baerii (Ab) IgM Y13253; bowfin, Amia calva (Ac)
IgM U12456; marbled lungfish, Protopterus aethiopicus (Pae) IgM
AF437724; human, Homo sapiens IgM 4467842; mouse, Mus musculus,
IgA AAB59662; human IgA2 546799; lungfish IgW AF437727; and
IgXlong clone 9 (8), using Clustal X version 1.8 (27). Phylogenetic tree
analysis was performed as described (28) using Clustal X version 1.8 and
1000 bootstrap cycles. The phylogram was drawn in Treeview version
1.6.6 (29) and labeled in Canvas version 9.0 (ACD Deneba Software,
Miami, FL).
Horn shark and nurse shark IgW Tm cDNA sequences were translated
into proteins using the EXPASY translate tool and aligned in Clustal W
version 1.8 (30) with Ig Tms from other vertebrate species using GenBank
accession numbers referenced in Campbell et al. (31) and as follows: human IgM X17115; mouse IgM P01873; Xenopus IgM X90768; Xenopus
IgY X90767; horn shark IgM X07781; Xenopus IgX CAA62293; channel
catfish IgD AF363448; mouse IgG1 P01869; mouse IgG3 P03987; mouse
IgG2a P01865; mouse IgG2b P01867; human IgG1 X52847; human IgE
A46485; mouse IgE X03624; Atlantic cod IgM X58871; channel catfish
IgM X52617; duck IgA CAC43282; human IgA2 M60194; human IgA1
AAA52745; mouse IgA K00691; horn shark IgW Tm2T7 P83978; nurse
shark IgW Tm1T3 P83981; nurse shark IgWshort clone 2-5 AY524296;
nurse shark IgW Tm clone 1E AY524297; nurse shark IgWshort clone 25E
AY531553-AY531554; nurse shark IgNAR clone 7A U18721; nurse shark
IgNAR NEP83977; horn shark IgW Tm7T7 P83979; horn shark IgW
Tm3T3 P83980; nurse shark IgM Tm 1E AY609247; nurse shark IgW
Tm6T3 P83982; nurse shark IgW Tm3C4 P83983.
The Journal of Immunology
1131
Table II. Primersa
Ig Class
Sequence
Priming Site
Anneal Temp
␻CH3 For
␻CH6 For
␻TM 1E For
␻TM 1E Rev
␻⬘ UT TM 1E Rev
␻SEC Rev
␻3⬘ UT SEC Rev
␻NEW12A FR1 For
␻12A CH1 Rev
␻CH6 For (38E)
␻CH6 Rev (38E)
␻HS FR1 For
␻HS CH1 Rev
␯CH2 (3– 4) For
␯CH5 (3– 4) For
␯TM (7A) Rev
␯3⬘ UT TM (7A) Rev
␯SEC (3– 4) Rev
␯3⬘ UTSEC(3– 4) Rev
␯FR1 VH (3– 4) For
␯FR3 VH (3– 4) Rev
␮HS VH For (3–5A)
␮HS VH Rev (3–5A)
␮VH FR1 For
␮VH FR4 (50S) Rev
␮CH1 Rev
␮TM For (35S)
␮3⬘ UT TMRev(35S)
IgW
IgW
IgW
IgW
IgW
IgW
IgW
IgW
IgW
IgW
IgW
IgW
IgW
IgNAR
IgNAR
IgNAR
IgNAR
IgNAR
IgNAR
IgNAR
IgNAR
IgM
IgM
IgM
IgM
IgM
IgM
IgM
5⬘-ACAGCAGTCTGTTCTGATCCCAGC-3⬘
5⬘-GGAGGCTGGAACTCGGGCAGT-3⬘
5⬘-GTGGTCCCCCCAAATGTGAAA-3⬘
5⬘-GGCCGGAACTGTTGGCGCGTT-3⬘
5⬘-CACAAGACCTAAGGAATCAGTC-3⬘
5⬘-GGAGTTAAAGCTTTCAG-3⬘
5⬘-TCGCACATGATCAGGGACACG-3⬘
5⬘-CCGAGTCAGTTGTGAAAAAGCC-3⬘
5⬘-TTAGAGCTTGTGTCACCG-3⬘
5⬘-GAGGTTAAGACTCACCCTCAA-3⬘
5⬘-GCTGTGGGATTTATTAATGCT-3⬘
5⬘-AATATCGTGTTGACCCAGCCC-3⬘
5⬘-CCTGCCTTGCAGTGGTAGAC-3⬘
5⬘-CAAATGGAACCAACTAAAATG-3⬘
5⬘-AAGGGGAGTGGTTCCAGCTTCGTT-3⬘
5⬘-AGTCATGATGGATAATGAACT-3⬘
5⬘-TCAGCTTGTAGCAGTTAAGTG-3⬘
5⬘-GGATTTAACAGTGTCGC-3⬘
5⬘-ATGTGACTATTGCGTGCAATGA-3⬘
5⬘-GAGCGAGTGGACCAAACGCC-3⬘
5⬘-ACCGCAACGATACGTGCCAC-3⬘
5⬘-GATGTCGTGTTGACTCAGCCA-3⬘
5⬘-AGTCACCGTCACCATGGTCCC-3⬘
5⬘-GAGATTACTTTGATTCAACCA-3⬘
5⬘-TGTAGTCACGGTCACCATGGT-3⬘
5⬘-ACGTCGGGGGAATAGTCCATCG-3⬘
5⬘-TCGATAGATCACACTTGGATT-3⬘
5⬘-TCGTAATTACCTCAATGATAT-3⬘
TAVCSDPS
GGWNSGS
VVPPNVK
NAPTVPA
64
64
64
64
64
48
66
52
52
56
56
64
64
56
64
58
58
50
62
64
64
62
62
50
56
64
53
53
NS NDPK For
NS NDPK Rev
NDPK
NDPK
5⬘-GGTAACAAGGAACGAACCTTC-3⬘
5⬘-CTCATAGATCCAGTCTTGGGC-3⬘
a
TESFNS
ESVVKK
GDTSSN
EVKTHPQ
SINKSHS
NIVLTQ
VYHCKAG
QMEPTKM
KGSGSSFV
SSLSIMT
SDTVKS
ERVDQTP
GGTYRCG
DVVLTQP
EITLIQP
TMVTTT
AMDYSPD
SIDHTWI
GNKERTF
AQDWIYE
64
64
For, forward; Rev, reverse.
until directly demonstrated as membrane-associated proteins
through biochemical characterization.
Inspection of cDNA clones from one animal revealed that IgW
mRNA is transcribed from at least two different loci in nurse
sharks, which are most divergent in the deduced CH2 domain (Fig.
1). The deduced amino acid identity between these two shark loci
for V (framework region of V domain (FR)1-FR3), CH1, and CH2
are 72, 61, and 46%, respectively. Skate IgWshort (previously
called IgX) is ⬃50% identical (amino acid) to either shark IgW
locus for each VH, CH1, and CH2 domain (data not shown). This
implies that the two loci diverged not long after the divergence of
sharks and batoids (skate) 220 million years ago. The deduced AA
identities for CH3-CH6 between the two shark IgW loci are much
higher (Fig. 1), indicating that those domains have been under
more stringent negative selection. Importantly, all cDNA forms of
IgW, including IgW Tm, IgW Sec, IgWshort Tm, and IgWshort Sec,
were encoded by each locus (or very closely related loci), implying
they are products of alternative splicing rather than from separate
loci encoding each form.
Alignment of the horn shark IgWshort Sec cDNA deduced protein sequences, isolated by differential screening of the horn shark
cDNA library, with the nurse shark IgWshort Sec cDNA-deduced
protein sequences identified the shark transcripts as orthologs to
the previously identified three-domain skate IgWshort cDNA having an unusual noncanonical (NC) Cys-rich Sec tail (9) (data not
shown and Fig. 2A). We detected transcription of only one horn
shark locus (or several closely related loci) with this NC Sec tail,
which contains seven Cys and is somewhat longer than the nurse
shark and skate NC Sec tails. Consistent with the presence of two
very different nurse shark IgW loci, two divergent NC Sec tail
cDNA sequences were identified that differ in length and Cys content. The NC Sec tail is dramatically distinct from canonical IgW/
IgNAR/IgM/IgA Sec tails, which are small, uniform in size, and
contain an invariant Asn-linked glycosylation site and only one
Cys in the penultimate position. In mammalian IgM and IgA both
the Cys and the glycosylation site are required for J-chain association (32–34). The deduced IgWshort NC Sec tail in all species
contains several Cys residues that could participate in either intraor interchain disulfide bonds (or even J-chain association) and one
potential Asn-linked glycosylation site.
Phylogenetic analysis demonstrates that the NC Sec tails diverge rapidly over evolutionary time (Fig. 2B). As described (Fig.
2A), nurse sharks (Gc) have two divergent loci for this NC tail,
whereas the horn shark (Hf) may have only one; note that the
divergence between the two nurse shark NC Sec tails is greater
than that between the canonical Sec tails of nurse shark and skate
(Re). Together, these results prove that IgWshort and its NC Sec tail
were present in the elasmobranch common ancestor. Unfortunately, IgWshort in the lungfish was only detected by Northern
blotting (5) and no cDNA sequence of its Sec tail was reported for
inclusion in this phylogenetic analysis.
Previously our laboratory identified H chain cDNA for both the
IgNAR Tm and Sec forms having six Ig superfamily (Igsf) domains through screening of an adult splenic cDNA library (19).
However, in another previous study examining IgNAR CDR3 diversification, serendipitously we identified Tm cDNA by RT-PCR
containing only three putative CH domains, suggesting that IgNAR, like IgW, underwent alternative splicing of its Tm mRNA
(12, 14). Thus, we further investigated the expression of IgW and
IgNAR Tm mRNA by RT-PCR of nurse shark epigonal organ,
spleen, and PBL (Fig. 3). Our strategy was to confirm: 1) IgNAR
Tm mRNA exists in two forms (CH3-Tm and CH5-Tm), and 2)
there is no Tm mRNA form of IgW having the same number of
Igsf domains as the Sec mRNA (CH6-Tm). IgW forward primers
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Primers
1132
DIVERSITY OF CARTILAGINOUS FISH Ig GENE RNA PROCESSING
Table III. RT-PCR products
IgW
IgNAR
Priming site
Full-length (bp)
Truncated (bp)
Fig. 3A row
CH3-Tm
CH3–3⬘ UT Tm
CH3-Sec
CH6-Sec
1281
1592
1245
183
636
947
trunc CH3-Tm
trunc CH3–3⬘ UT Tm
Full-length CH3-Sec
Full-length CH6-Sec
Cartilaginous fish IgW, IgNAR, and IgM Tms contain the
conserved Ag receptor motif (CART)
The deduced Tm regions of horn shark and nurse shark IgW,
IgWshort, IgNAR, and IgM cDNA clones were aligned with each
other and with other vertebrate Ig Tms (Fig. 4). Shark Tms
conform to a highly conserved motif in Ag-receptor phylogeny,
CH2-Tm
CH2–3⬘ UT
CH5-Tm
CH5–3⬘ UT
CH2-Sec
CH2–3⬘ UT
CH5-Sec
CH5–3⬘ UT
Tm
Tm
Sec
Sec
Full-length (bp)
Truncated (bp)
Fig. 3B lanes
1257
1585
243
569
1170
1300
183
307
586
914
1–2
3–4
5–6
7–8
9–10
11–12
13–14
15–16
consisting of three components: an extracellular acid-rich spacer
region, a hydrophobic membrane-spanning region, and a short
cytoplasmic tail (18, 31). The extracellular regions of the IgW and
IgNAR Tm forms are acidic, but as is true of Tm forms in other
vertebrates, the sequence and length of this region are variable
(31). In contrast, the Tm hydrophobic region is well conserved in
sequence and size throughout phylogeny. The short cytoplasmic
tail is also conserved, as was shown previously for horn shark IgM
Tm cDNA (18), and now for IgW and IgNAR Tm cDNAs. The
putative cytoplasmic tail of IgNAR Tm cDNA retains a polar
residue as the last amino acid, substituting Asn for the more typical
Lys. The putative IgW Tm cytoplasmic tail is composed of four
amino acids, with a substitution of two Gln for the ultimate Lys. In
mammalian Ig, the Lys residues are thought to tether the receptor
at the plasma membrane, and are important for signaling, Ag
presentation, and internalization of the Ig receptor (31); the terminal residues in IgW and IgNAR cytoplasmic tails are somewhat
more similar to mammalian IgE and IgA than to IgM, suggesting
a mechanism for differential signaling.
Deduced nurse shark IgM Tm precisely conserves the residues
shown to be important in the mammalian hydrophobic region.
Within this region, Thr, Ser, and Tyr residues are required for
mammalian Ig␣/Ig␤ coreceptor association at specific positions (ⴱ,
Fig. 4) (31). These residues are conserved in all surmised shark Ig
Tms suggesting they could interact with these essential coreceptors. Residues in the Tm region form ␣-helical secondary structures having hydrophilic and hydrophobic portions. Fig. 5 compares the shark IgNAR and IgW Tm regions to the CART using
helical wheel plots (refer to Fig. 4) (31). All shark Ig isotypes
contain deduced residues in this Tm region that match the conserved motif in both their positions in the helix and residue qualities. Together, these data demonstrate that the surmised shark
IgW, IgNAR, and IgM Tms preserve the conserved Ag receptor
Tm motif, and thus their putative proteins may associate with an
Ig␣/Ig␤-like coreceptor and signal through their C-terminal regions. Additionally, these deduced shark Ig proteins present a conserved ␣-helical conformation at the membrane interface with features shown to be important for structure and signaling in
mammals.
IgW and IgNAR Tm mRNAs are highly expressed in the spleen
and PBL, and enriched in PBL
The tissue distribution of IgW and IgNAR mRNA in an adult nurse
shark was examined by Northern analyses with probes specific for
either the Tm forms or all forms (Fig. 6, refer to Table I for
probes). Note that the Tm forms are generally found at much lower
levels than the Sec forms (15), and thus the Tm forms are difficult
to detect with probes that hybridize to both forms (e.g., compare
the PBL lane for IgW Tm and IgW Sec, Fig. 6, A and B: the Tm
form at 2.3 kb (band 2 in Fig. 6A) is hardly visible in Fig. 6B using
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were designed for the deduced CH3 or CH6 domains and reverse
primers for the deduced Tm, 3⬘ untranslated region (UT) Tm, or
Sec regions (Fig. 3A, refer to Table II for primers; expected PCR
sizes in Table III). Only PCR products of 636 bp (CH3/Tm, Fig.
3A, first row) or 947 bp (CH3/3⬘ UT Tm, Fig. 3A, second row) for
IgW Tm cDNA were produced under various PCR conditions with
several individuals, representing the CH4-Tm spliced product (note
that the PCR fragments were confirmed by sequencing). Other
primer sets that could amplify the CH6 and Tm or 3⬘ UT Tm
mRNA produced no bands (Fig. 3A, rows 5 and 6), confirming the
absence of an IgW Tm transcript with seven putative Igsf domains.
Other controls showed that only full-length products (encoding
seven domains) of 1245 and 183 bp were amplified with CH3 and
Sec primers, and CH6 and Sec primers, respectively (Fig. 3A, rows
3 and 4). Thus, we confirmed that there is no detectable mRNA
encoding an IgW Tm form with seven-Igsf domains in primary and
secondary lymphoid tissues.
RT-PCR of the same tissue mRNA was done for the IgNAR Tm
study (Fig. 3B). As mentioned, previous data suggested that IgNAR Tm mRNA occurred in two sizes, one with five putative
constant domains and another lacking the two C-terminal domains.
Thus, two bands were expected to be amplified using the primer
pairs CH2/Tm (Fig. 3B, lane 1) or CH2/3⬘ UT Tm (Fig. 3B, lane 3,
refer to Table III). Indeed, two bands were amplified with these
primer sets, representing the six and four domain-encoding cDNAs,
respectively (Fig. 3B, black arrows in rows 1 and 3). The band
representing the smaller form was always more intense, which
either suggests that it is preferentially amplified or is truly expressed at higher levels; the fact that an intense band representing
the high m.w. product in Fig. 3B, lanes 1 and 3, was amplified with
CH5/3⬘ UT Tm primers favors the former possibility (Fig. 3B, lane
5). Tm exons for the mRNA encoding the deduced five-CH domain
form are spliced to the cryptic splice site within the exon encoding
CH5, while the form with three CH domains is spliced to the Tm
exons at the canonical splice site that flanks the exon (Fig. 3C).
Primer pairs for CH2 or CH5 and Sec or 3⬘ UT Sec verified that
IgNAR Sec mRNA is present only as a six-domain form, consistent with previous molecular and biochemical data (Ref. 12, Fig.
3B, lanes 9 –16). In summary, IgNAR Tm mRNA is found in two
forms in primary and secondary lymphoid tissues, one form having
the same number of putative CH domains as the Sec form (unlike
IgW Tm); furthermore, unlike IgW Sec, we find mRNA of only
one size encoding the deduced IgNAR Sec.
Priming site
The Journal of Immunology
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FIGURE 1. IgW and IgWshort Tm and Sec cDNA
forms are probably alternatively spliced transcripts. The
various forms of IgW were aligned from cDNA clones
(eight clones for locus 1 and eight clones for locus 2) for
IgW Sec (LS), IgW Tm (LTM), IgWshort Sec (SS), and
IgWshort Tm (STM) obtained from adult nurse shark (Y)
and compared with the original adult nurse shark IgW
Sec (U51450; Ref. 6); note that the original IgW clone
was not derived from the same shark. The major amino
acid differences between the VH, CH1, and CH2 domains
indicate that at least two separate IgW loci are expressed.
The few amino acid differences detected within the same
family could either be derived from somatic changes or
multiple closely related IgW loci. Refer to Fig. 8 for
diagrammatic views of the various putative forms of
IgW. GenBank accession numbers are as follows: clone
1-1 AY524294, clone 1-2 AY524282, clone 1-3
AY524289, clone 1-4 AY524279, clone 1-5
AY524277(5⬘end), AY524278 (3⬘end), clone 1-6
AY524286, clone 1-7 AY524280, clone 1-8 AY524283,
clone 2-1 AY524291, clone 2-2 AY524288, clone 2-3
AY524284, clone 2-4 AY524287, clone 2-5 AY524296,
clone 2-6 AY524285, clone 2-7 AY524281, clone 2-8
AY524290.
1133
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DIVERSITY OF CARTILAGINOUS FISH Ig GENE RNA PROCESSING
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FIGURE 2. Comparisons of the IgWshort Sec tails among elasmobranch species. A, IgWshort NC Sec tails from horn shark (Hf) and nurse shark (Gc) were
aligned with that of skate (Re) IgWshort. Canonical Sec tails from cartilaginous fish IgNAR, IgW, IgM, and lobe-finned fish IgM and IgW were also aligned
with mammalian IgM and IgA. A putative N-linked glycosylation site (bold asterisk and underlined) and multiple Cys residues (bold) are present in all of
the NC Sec tails. The canonical tails for IgW and IgM are highly conserved in cartilaginous fish; the invariant glycosylation site and Cys are noted (bold
asterisk and underlined). Slashes (/) indicate gaps added for alignment purposes (Hf, H. francisci; Gc, G. cirratum; Re, R. eglanteria; Rp, R. productus;
Ab, A. baerii; Hc, H. colliei; Ac, A. calva; Pae, P. aethiopicus; Mm, M. musculus; Hs, H. sapiens. B, Phylogenetic trees for the Sec tails derived from the
sequences in A. The trees were rooted using the canonical tail from human (H. sapiens, Hs) IgM. Abbreviations are the same as in A.
the probe that recognizes both forms). IgW and IgWshort Tm
mRNAs were expressed in a restricted pattern, present only in the
spleen, pancreas, PBL, and at low levels in epigonal organ (Fig.
6A). Bands 2 and 3 correspond to the putative five and three domain IgW Tm forms, respectively; band 1 has not been detected in
cDNA library screens or by RT-PCR. IgW Sec expression was also
restricted, with high expression found in the spleen, epigonal organ, and testes. Low levels of IgWshort were observed in multiple
tissues, including the liver, gill, kidney, esophagus, and testes and
enriched expression in the pancreas of this animal. A higher m.w.
IgW Sec transcript (3.6 kb-band 1, Fig. 6, A and B) was detected
mainly in the spleen; this transcript has never been identified in multiple library screenings, and may be unspliced heterogeneous RNA as
seen in nurse shark IgM expression (M. F. Flajnik, unpublished results), or perhaps another form of IgW mRNA resistant to cloning.
Previously, we have shown that the epigonal organ, a bone marrow
The Journal of Immunology
1135
equivalent in cartilaginous fish, functions as a reservoir for Sec Igs
(21, 35), a situation similar to that of plasma cells residing in mammalian bone marrow. Thus, we expected to find IgW Sec mRNA
expression there as well. As expected, based on the RT-PCR experiment (Fig. 3B) and previous biochemical experiments (12), IgNAR
Sec was present as a single mRNA species and transcribed in most
tissues (Fig. 6D), an expression pattern largely overlapping with IgM
Sec (Fig. 6E); only IgM Tm was expressed in the thymus.
In summary, Tm mRNA of all isotypes is enriched in the spleen
and especially in the PBL (Fig. 6, A, C, and E). IgNAR and IgM Sec
mRNAs have a similar, broad tissue distribution. In contrast, IgWshort
Sec mRNA is expressed primarily in the spleen, pancreas, epigonal
organ, and at low levels in several other tissues; IgW Sec mRNA
expression is seen in these same tissues as well as in the testes.
spleens of six adults and five pups to determine whether expression
of this isotype is universal. Among the six adults investigated,
IgWshort mRNA was strongly expressed in four animals and hardly
at all in the others (Figs. 6 and 7, data not shown). Among very
young animals, IgW and IgWshort mRNA expression varied between individuals ranging from high to very low. We conclude that
both forms of IgW mRNA can be expressed early in ontogeny, at
least during late embryonic development and at birth. We also
conclude that, for unknown reasons, IgWshort can be expressed at
very different levels in individual nurse sharks, providing an explanation for not detecting this form of IgW mRNA previously in
sharks.
Individual nurse sharks express IgWshort mRNA differentially in
secondary lymphoid tissue
Multiple forms of Ig isotypes in the cartilaginous fish
Because IgWshort was believed not to exist in sharks in previous
studies (2, 4, 7), we investigated Ig mRNA expression in the
Discussion
Fig. 8 shows our current understanding of cartilaginous fish Ig at
the cDNA and/or protein levels. The five-domain IgM is clearly
the most abundant serum Ig, present in 19S and 7S forms at nearly
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FIGURE 3. IgW and IgNAR Tms are alternatively spliced in primary and secondary lymphoid tissues and in PBL. The epigonal organ, spleen, and PBL
of an adult nurse shark were examined for IgW and IgNAR Tm and Sec expression by RT-PCR; primers for PCR noted in Table II and expected sizes of
PCR products in Table III. A, IgW Tm is alternatively spliced. PCR positive controls were amplified products using plasmid cDNA templates. In row 1,
primer pair CH3/Tm amplified only the alternatively spliced transcript of 636 bp in each tissue, i.e., the five-domain form. In row 2, primer pair CH3/3⬘
UT Tm gave the same results of a truncated PCR product of 947 bp. Rows 3 and 4 that demonstrated IgW Sec is expressed as a full-length transcript, i.e.,
the seven-domain form, using primer pairs CH3/Sec and ␻CH6/Sec, which amplified 1245- and 183-bp products, respectively. Rows 5 and 6 show that the
primer pairs CH6/Tm and CH6/3⬘UT Tm amplified no bands of the expected size, confirming that there is no expressed IgW Tm form with seven Igsf
domains. B, IgNAR Tm is alternatively spliced to produce four- and six-domain Tm forms. Amplified products from tissue RNA are shown in the odd lanes
and positive controls (cDNA inserts in plasmids) in the even lanes. In lane 1, two PCR products (arrows) were amplified using primer pair CH2/Tm that
represent the full-length (1257 bp) and truncated (586 bp) IgNAR Tms (arrows). The same results were obtained in lane 3 using primer pair CH2/3⬘UT
Tm. In lanes 5 and 7, using primer pairs CH5/Tm and CH5/3⬘UT Tm full-length, i.e., six-domain, IgNAR Tm expression was verified. IgNAR Sec is
expressed only as a full-length form, shown in lanes 9 (CH3-Sec), 11 (CH3-3⬘UTSec), 13 (CH5-Sec), and 15 (CH5-3⬘UTSec). C, Sequences at the splice
junctions of the four-domain (CH3/Tm) and six-domain (CH5/Tm) IgNAR Tm products. The Tm exon splices to the cryptic splice site in the CH5 exon and
to the canonical splice site in the CH3 exon (3).
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DIVERSITY OF CARTILAGINOUS FISH Ig GENE RNA PROCESSING
equivalent levels in adults (36). The deduced Tm form of the IgM
H chain has five domains, as is seen in most vertebrates. Many
IgM loci exist in all cartilaginous fish, and one gene cluster diversified through formation of a germline-joined H chain locus encoding the four-domain IgM1gj, so far found only in nurse sharks
(2, 21). IgM1gj protein may have a function in early development
as it is secreted in high amounts at birth. No Tm cDNA has been
identified for this isotype and one may not exist. Results reported
in this paper, together with previous data in batoids (8 –10), the
frill shark (11), and lungfish (5) reveal that IgW has been selected
to diversify its size using alternative mRNA splicing for two shortened Tm forms and a short Sec form, IgWshort. In contrast to IgW,
only the IgNAR Tm form can be present in a truncated form and
there are no multiple Sec forms. The structures, tissue distribution,
and levels of somatic mutation differ among these three major Ig
classes, strongly suggesting that each isotype has a distinct effector
function, as shown for mammalian Ig classes (37). Thus, these
ancient animals have a much greater complexity of Ig than previously believed, and it is predicted that the isotypes, and all their
different forms, fulfill particular needs for their adaptive immune
systems.
Teleology for the short IgNAR and IgW Tm mRNA forms
As described, bony fish splice the putative TM1 exon to the ␮CH3
donor splice site producing a deduced membrane-bound IgM that
is smaller by one domain (3). Tm IgW and IgNAR cDNAs are
encoded by alternatively spliced products that result in putative
truncation by two domains at the C terminus (and a second form of
IgW Tm with only three domains, Fig. 8). It is difficult to fathom
the functional reason(s) for the shortened Tm cDNA forms in
teleosts. Teleost IgM Sec is unique among vertebrates in that it is
tetrameric and assembles its quaternary protein structure in diverse
ways through variable disulfide bonding between the ␮ monomers
and half-mers (38). Perhaps a truncated IgM Tm on the bony fish
B cell surface reduces or inhibits this structural diversity to ensure
proper BCR signaling. For IgNAR, and by inference IgW, we
propose a simple explanation for the alternative splicing suggested
by our previous molecular and structural studies. Our previous
work found IgW cDNA is homologous to IgNAR cDNA in the last
four putative constant domains, i.e., these two isotypes shared a
common ancestor ⬃220 million years ago, before the batoid
(skate, ray) and shark divergence (6). Our structural study of
IgNAR Sec proteins isolated by a specific mAb identified a highly
flexible region between the third and fourth CH domains that were
shown to bend up to 90° by immunoelectron microscopy (Ref. 13;
inset, Fig. 8). In the putative IgNAR Tm form with three CH
domains (Tm-2, Fig. 8) the alternative splicing would result in the
removal of this bend region. The cDNA homology at their C
termini between the two isotypes makes it likely that the same
bend will be found in IgW Sec and alternative splicing to produce
the truncated IgW Tm cDNA (VH-CH4) would result in its removal. We have preliminary evidence that this “bend” in IgNAR
Sec proteins may be sensitive to proteolysis because Western
analysis of IgNAR under denaturing conditions produces a smaller
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FIGURE 4. IgW and IgNAR Tms are canonical. Horn shark and nurse shark IgW, IgWshort, and IgM, and nurse shark IgNAR Tm deduced residues were
compared with Tms from all classes of vertebrate Igs and aligned in ClustalW. Shark IgW and IgNAR Tms contain the CART, composed of an acidic
extracellular spacer region, a Tm region consisting of hydrophobic and polar residues, and a short cytoplasmic tail important for receptor signaling (31).
All of the Tms contain the essential polar residues threonine (T), serine (S), and tyrosine (Y) (bold asterisks) in positions required for Ig ␣␤ association.
Slashes (/) denote gaps added for alignment purposes and dashes (-) indicate residues identical to human IgM.
The Journal of Immunology
1137
band the size of two CH domains recognized by the anti-IgNAR
tail region specific mAb (H. Dooley and M. F. Flajnik, unpublished observations) which suggests it may be disadvantageous to
express the “full-length” molecules at the cell surface. These
preliminary data requires protein sequencing to verify the Western
results. Evidence to date suggests that, despite their many clusters
of Ig genes, each elasmobranch B cell expresses only one IgH locus
(35, 39). Thus, if IgM, IgW, and IgNAR Tm are expressed clonally
in each B cell, the shorter forms may limit proteolysis and permit
signaling to occur in a similar fashion for all isotypes (at least
regarding cross-linking of the receptor). Differential signaling
among the isotypes may be, in part, indicated by amino acid
differences in the cytosolic tails of IgW and IgNAR Tm, as
compared to IgM (refer to Fig. 4). In contrast, Sec forms are
composed of seven (IgW), six (IgNAR), or five (IgM) Igsf domains and this size may impact the type of effector responses
generated when all three isotypes are induced in an Ag-specific
response, i.e., if FcRs exist for all three isotypes on one type of
hemopoietic cell, the longest one might “win” in the induction of
a particular effector outcome.
Role of IgWshort Sec in immune responses
It has been more than a decade since skate IgWshort cDNA (formerly called IgXshort) was discovered and we now report that its
ortholog in sharks is IgWshort cDNA, a miniature version of IgW.
To date, IgWshort proteins have not been isolated in skates or
sharks thus this discussion is based on mRNA molecular data. Like
the deduced skate IgWshort, the shark deduced IgWshort consists of
a VH-CH1-CH2-NC Sec tail. It can be expressed in late-term gestation nurse shark pups, just before birth and thereafter during
ontogeny, and thus it does not appear to be developmentally regulated as seen for IgNAR and 7S IgM (21, 35). IgWshort is most
highly expressed in the spleen, the only true secondary lymphoid
tissue in cartilaginous fish. The high IgW levels in the spleen are
of interest because that tissue is immature in newborn pups, colonized by IgM-bearing B cells yet lacking T cell zones and dendritic cells (35); consequently neonatal spleen lacks the environment necessary for Ag-specific responses. One possibility is that
the IgW expressed by young animals is encoded by germlinejoined loci, yet this is unlikely as no germline-joined IgW locus
has been discovered in nurse sharks after extensive library screens
of adult and (especially) neonatal libraries. A second possibility is
that IgW-expressing B cells develop earlier than B cells expressing
other Ig isotypes in utero and receive the necessary (perhaps Tindependent) signals to become activated and secrete. This second
choice can be tested once specific mAbs have been made that will
identify the Tm and Sec forms of IgW and IgWshort.
Variable IgWshort and IgW expression between individuals may
be indicative of the amount or type of Ag to which these individuals have been exposed, and because IgW is secreted in significantly lower amounts than IgM and IgNAR, changes in its expression levels may be easier to detect (6, 21). In unimmunized adults
(exposed to native ocean pathogens), IgWshort Tm and Sec forms
are expressed in a novel site, the pancreas. As in mammals, the
shark pancreas has exocrine (digestive enzymes) and endocrine
(regulation of carbohydrate, protein, and lipid metabolism) functions (40, 41). Thus, in addition to secretion of digestive enzymes
into the lumen of the intestine, sharks may secrete IgWshort as a
GALT-type protection. Besides the pancreas, low levels of IgWshort
were observed in multiple tissues; perhaps due to its small size and
potential access to extracellular spaces, IgWshort-secreting cells
may be enriched in these areas. There is a precedent in other vertebrates for expression of a truncated form of Ig: in ducks a short
form of IgY exists that is derived by alternative splicing (42). It has
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FIGURE 5. Hydrophobic and hydrophilic features of conserved Tm regions are maintained in IgW and IgNAR Tms. The Ig Tm region is based on
conserved residues identified among vertebrate Ig classes (31). The IgNAR and IgW Tm residues starting after the tryptophan (W) (refer to Fig. 4) and
ending one residue before the cytoplasmic tail were compared with the conserved Tm motif using a helical wheel, which displays ␣-helix amino acid
residues at 100° intervals or 3.6 residues per 360°. Note that the amino acid characteristics in the shark Ig Tms form a conserved ␣-helical structure.
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DIVERSITY OF CARTILAGINOUS FISH Ig GENE RNA PROCESSING
been suggested that this truncated form has been selected to limit
inflammatory reactions and thus may be more of a neutralizing Ab
(43). This is likely to be true in the cartilaginous fish as well, and
the hit-and-miss nature of finding IgWshort Sec forms in different
individuals provides an explanation for the previous proposal that
sharks only had the long form of IgW Sec. Like in ducks, we
suggest that the presence/absence of IgWshort is a consequence of
the types of ongoing immune responses at the time of analysis.
Have all of the cartilaginous fish isotypes and their different
forms been discovered?
Fig. 8 shows that the cartilaginous fish have a large assortment of
Ig isotypes, however, there may be more. We were unable to clone
two forms of IgW mRNA (one Tm and one Sec) that may encode
high m.w. proteins that we have detected from time to time in
immunoprecipitation assays (M. F. Flajnik and H. Dooley, unpublished data). Thus, we must remain open to the possibility that
there are other types or forms of shark Ig. Interestingly, despite the
FIGURE 7. IgWshort is differentially expressed among nurse shark individuals. Northern analysis of IgWlong and IgWshort splenic expression in
various aged nurse shark individuals; adult (A) and neonate pup (P). A, An
IgW VH probe reveals that IgWlong is expressed by all individuals, regardless of age, with lower levels observed in young animals. IgWshort expression is more variable, ranging from high in adults 3 and 4, to very weak in
adult 1 and pup 4. Autoradiogram exposure at 3 days. B, An IgNAR VH
probe shows that IgNAR is expressed abundantly in the mature individuals,
and poorly in immature pups, as previously described (21, 35). Autoradiogram exposure at 1 day. C, IgM expression revealed with an IgM VH probe.
Note that adult 3 has lower levels of IgM, yet is enriched for IgW and
IgWshort expression (refer to A). Autoradiogram exposure at 4 h. D, NDPK
probe-NDPK loading control. Autoradiogram exposure at 4 h. E, An IgW
VH probe reveals further that all individuals regardless of age had highest
expression of IgW in the spleen. Pups preferentially express IgWshort. Note
that the three pups in E were from a different family than the two pups in
A–D. Epi, epigonal organ; Spl, spleen.
great evolutionary distance between different groups of elasmobranchs (up to 220 million years, two to three times as long ago as
the last common ancestor of placental mammals); all of the isotypes except IgM1gj have been identified to date in all of the different species so far examined.
Finally, are the different Tm and Sec forms truly derived by
alternative splicing? The evidence presented here and from previous work in the skate (8) strongly suggest that all of the different
forms can be encoded by a single locus. However, especially for
IgW, the cDNA clones that we have isolated may have been derived from very similar loci, and future studies are required to
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FIGURE 6. Expression of IgM, IgW and IgNAR. Northern analyses of
one adult nurse shark to examine the tissue distribution of IgW/IgWshort
Tm and Sec, and IgNAR Tm and Sec. Refer to Table I for probes. A, An
IgW Tm probe reveals three bands; the two bottom bands (nos. 2 and 3) at
2.6 and 1.3 kb represent the five and three Igsf domain forms, and the upper
band (no. 1) has been resistant to cloning. Autoradiogram exposure 13
days. B, An IgW VH probe reveals four bands of IgW Sec. The band at 2.6
kb (no. 2) represents the seven-domain form (6) and the 1.2-kb band (no.
4) the three-domain form. No. 3 indicates cross-hybridization of the IgW V
probe with IgM Sec, which is present in very high amounts in the spleen
(see E). The highest m.w. band (no. 1) has been resistant to cloning. Autoradiogram exposure at 10 days. C, An IgNAR Tm probe reveals two
bands in spleen and PBL. Autoradiogram exposure at 9 days. D, An IgNAR
VH probe reveals that IgNAR Sec mRNA is a single transcript with broad
tissue distribution. Film exposure at 6 days. E, An IgM VH probe reveals
very high expression in spleen and a broad tissue distribution. IgM Tm can
be seen as a 2.6-kb band in PBL and thymus (swamped out in the spleen
by the Sec form). Autoradiogram exposure at 1 day. F, The housekeeping
gene nucleoside diphosphate kinase (NDPK) was used as an RNA loading
control. Autoradiogram exposure at 1 day. Hrt, heart; Sto, stomach; Mus,
muscle; Spl, spleen; Int, intestine; Pan, pancreas; Liv, liver; Gil, gill; Rec,
rectal gland; Thy, thymus; Kid, kidney; Brn, brain; Epi, epigonal organ;
PBL; Eso, esophagus; Tes, testes; Olf, olfactory bulb.
The Journal of Immunology
isolate all of the IgW gene clusters from individual animals to
confirm the splicing hypothesis.
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FIGURE 8. Cartilaginous fish Ig repertoire. Current molecular/protein
models of Ig isotypes identified thus far in cartilaginous fish. See text for
details.
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