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Dairy Science Group, AgResearch, Ruakura Research Centre, Hamilton, New Zealand
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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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200 days, during which
milk composition and the pouch young’s behavior changes. In early
lactation (first 80 days), the marsupial young is confined to the pouch
where it remains attached to the nipple, receiving milk of unique
protein, carbohydrate, mineral, and fat composition (4, 5, 6). During the
switch phase (days 80–120 of lactation), the milk composition changes
to become similar in composition to that of eutherian mammals. With the
change in milk composition, growth of the pouch young increases, and
they undergo changes such as fur growth, their activity increases,
suckling is more intermittent (after day 80), and they begin to leave
the pouch (after day 115). At birth, eutherian young are immunologically naive and they receive immune protection by ingestion of Igs secreted into the colostrum until they are capable of mounting their own immune response. In mammals such as humans, where IgG is transferred predominately via the placenta, IgA is the major Ig transferred via the colostrum. In contrast, when little or no IgG is transferred before birth, as seen in the cow, more IgG than IgA is transferred in the colostrum (7). For marsupials, little is known about Ig transfer via the placenta or mammary gland. In the newborn of several marsupials, no Igs were detected in their serum until they had suckled for the first time, suggesting that in these species there is only postnatal transfer of Igs (8, 9, 10). In contrast, Igs have been detected in the serum of newborn of the tammar wallaby (Macropus eugenii) before suckling (11). The transfer of Igs in utero has not been investigated for the possum. It has been shown that newborn possums are unable to mount a humoral response for at least 14 days after birth (12). Thus postpartum transfer of both IgG and IgA is likely to be important for immune protection of the possum young. A study of three brushtail possums demonstrated that Igs ingested by 50- and 98- but not 145-day-old pouch young were subsequently detected in their serum (13). This suggests that, although older pouch young can mount their own immune response, they remain receptive to the passive immunity offered by their mothers milk, at least into the switch phase of lactation.
Passively transferred IgA functions to protect mucus membranes
(respiratory and gastrointestinal) of the newborn. In eutherian
mammals, lymphocytes associated with mammary gland epithelium secrete
IgA as a dimer joined by the J chain (14). Dimeric IgA is transported
across the epithelial cells into the milk by the polymeric Ig receptor
(pIgR)4 and is released at the
apical membrane by proteolytic cleavage of the extracellular domain
(secretory component (SC)) (15). It has been suggested that the SC
protects dimeric IgA from proteolytic cleavage in mucosal environments
(16). The possum IgA heavy chain (C
) DNA sequence was published
recently by Bevlov et al. (17).
Immune transfer to the possum neonate via the milk has not been studied
in great detail. In this work, we have cloned and characterized the
first marsupial homologues of the pIgR and J chain genes. Analysis of
the expression pattern of the pIgR, J chain, and C mRNA suggests
that there are two stages of increased IgA transfer during lactation in
the possum, after birth and during the switch phase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mammary gland total RNA. Mammary glands were collected as described previously (6), and the date of lactation was based on the size of the pouch young (18). The frozen mammary tissues were ground under liquid nitrogen by mortar and pestle, and RNA was extracted using the guanidinium acid phenol chloroform method (19) with the modification of reducing the recommended amount of tissue per volume of reagent by one half.
White blood cell total RNA. RBC were lysed by the addition of 12 ml of lysis solution (150 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA) to 4.5 ml of heparin-treated possum blood, incubating on ice for 20 min. White blood cells were harvested by centrifugation and washed with 1x PBS before RNA isolation as described above.
RT-PCR and 5' rapid amplification of cDNA end (RACE)
Random or oligo-dT primed cDNA was generated using the Superscript preamplification system (Life Technologies, Gaithersburg, MD) as per the manufacturer’s instructions. PCR was performed using Platium Taq (Life Technologies) or Taq DNA polymerase (Boehringer Mannheim, Mannheim, Germany). The 5' RACE was performed as described by Life Technologies using Superscript II reverse transcriptase and RNase H from Life Technologies and terminal transferase kit and RNase T1 from Boehringer Mannheim. Primer-specific cDNA was purified using the BRESAclean DNA purification kit (Bresatec, Thebarton, Australia). Upstream primers, abridged anchor primer and abridged universal amplification primer, used in the PCR reactions are as described by Life Technologies. All PCR products were cloned into pGEM-Teasy (Promega, Madison, WI), and at least two clones of each were DNA sequenced.
pIgR. RT-PCR was performed with 5' primer (GAA TTC CTA GCA CCA RTA CCA NCC YTC) and 3' primer (GGA TCC TAY TGG TGY AAR TGG). PCR conditions were annealing at 37°C for 3 cycles, then 55°C for 27 cycles with 60-s extensions. The 5' RACE was performed with GSP1 (CTG GCA TAG AAA CTT GG), GSP2 (AGG GCA CAG TTC AAG GTC AC), and GSP3 (AGG CTC CAG CAA CTT TAA GTC). PCR conditions for PCR1 were annealing at 50°C for 10 cycles then 55°C for 20 cycles with 4-min extensions, and for PCR2 were annealing at 57°C for 30 cycles with 2.5-min extensions.
J chain. RT-PCR was performed with 5' primer (GGA TCC GYT GAY AAY AAR TG) and 3' primer (GGA TTC CTA RTC AGG RTA), annealing at 37°C for 4 cycles, 45°C for 4 cycles, then 50°C for 26 cycles, with 90-s extensions. RT-PCR analysis of J chain mRNA expression was performed with 5' primer (GAG GCA AGA TGA AGA GAT CT) and 3' primer (GTG CAG TTT CAA GAT GGA AG). PCR conditions were annealing at 58°C for 25 cycles with 80-s extensions.
IgA. RT-PCR was performed with 5' primer (GAG GCA AGA TGA AGA GAT CT) and 3' primer (GTG CAG TTT CAA GAT GGA AG) with four PCR cycles each at 65°C, 60°C, 57°C, 53°C, and 50°C, then 10 cycles at 60°C with 2-min extensions. For 5' RACE, primers GSP1 (CAA GCA GCC TAG GAC TA), GSP2 (AGT CTC CGA GTA GAA TGA G), and GSP3 (GGA GAC AGT CAC CAT G) were used as described for the pIgR.
Library screening
The EcoRI-EcoRI DNA fragments from clones containing pIgR and J chain RT-PCR products were purified and used to generate 32P-radiolabeled probes with the Rediprime random primer labeling kit (Amersham, Aylesbury, U.K.). These were used to screen the possum early lactation mammary gland cDNA library (20) using methodology previously described (21). Recombinants were purified to single plaques and the internal plasmid (pBS; Stratagene, La Jolla, CA) was excised with ExAssist helper phage (Stratagene) following recommended procedures. The resulting plasmids were characterized by DNA sequencing.
DNA sequencing
DNA sequencing reactions were conducted by the DNA Sequencing Unit (University of Waikato, Hamilton, New Zealand) using an automated DNA sequencer (Applied Biosystems, Foster City, CA). DNA sequence data was collated using the SEQMAN II program of the Lasergene software package (DNASTAR, Madison, WI).
Northern blotting
Total RNA (6 µg) was denatured in the presence of formamide and formaldehyde and then resolved by gel electrophoresis in 1.5% agarose, 0.6 M formaldehyde gels (22). RNA was transferred to Hybond N membrane (Amersham) by capillary blotting. Blots were hybridized overnight at 55°C with 32P-radiolabeled cDNA probes in Church and Gilbert hybridization solution (22) following 30 min prehybridization. Blots were washed in 2x SSC/0.1% SDS at 55°C, then 1x SSC/0.1% SDS for 20 min each. Blots were exposed to Kodak XAR-5 film (Rochester, NY) at -80°C with two intensifying screens. The possum 18S rRNA probe is an unpublished DNA fragment (400 bp) amplified by RT-PCR (J. Demmer, GenBank AF089722).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 156-bp DNA fragment corresponding to the possum pIgR was
amplified by RT-PCR using mammary gland total RNA (28 days lactation)
and oligonucleotide primers designed to conserved regions of the pIgR
coding sequence. This fragment was used to screen an early lactation
possum mammary gland cDNA library, and 12 positive cDNA clones were
isolated. Of these, three recombinants containing different-sized
inserts were characterized by DNA sequencing. All three recombinants
contained pIgR sequence, and the DNA sequence of the largest cDNA clone
(2066 bp) was determined for both DNA strands. This recombinant
contained the majority of the coding sequence (from nt 782; Fig. 1
). The remainder of the coding and 5'
untranslated sequences were obtained by the 5' RACE procedure. The
possum pIgR is a 733-aa polypeptide including the 17-aa signal peptide.
It shares 44–48% amino acid identity to human, murine, bovine, and
rat pIgR sequences, which is lower than that observed between these
species (56–80%) with the exception of the rabbit pIgR sequence,
which has low homology with the possum pIgR (40%) as well as eutherian
pIgR sequences (43–47%).
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). The 23-aa residue polymeric Ig-binding
domain, located in the first Ig-like domain, is involved in the initial
noncovalent interaction with polymeric Ig (23). In the case of dimeric
IgA, this is followed by the formation of a disulfide bridge between
one of the C chains and the fifth Ig-like domain of the pIgR (24).
In the possum pIgR, both the pIg-binding domain (Fig. 2A) and the cysteine residues
involved in the formation of the covalent interaction are conserved
(Fig. 1, circles). This suggests that the mechanism for receptor-Ig
interaction has been conserved in marsupials.
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B, boxed region). It has been
suggested that this region may be involved in transducing a signal,
generated by ligand binding, to the cytoplasmic membrane leading first
to endocytosis and then transcytosis of the receptor to the apical
membrane (25). The region of lowest sequence conservation was in the
linker region (domain VI; Fig. 1) connecting the Ig-like and
transmembrane domains. This region is the most variable between all the
pIgR sequences with respect to size and sequence (25).
The pIgR cytoplasmic domain contains signaling sequences for
basolateral targeting, endocytosis, and transcytosis (26, 27), and this
region of the possum pIgR is highly conserved with eutherian sequences
(68–78% identity). Signals required for basolateral targeting of the
de novo pIgR reside in a region proximal to the transmembrane domain.
Three amino acid residues have been previously defined as being
important for basolateral signaling in the rabbit pIgR (Fig. 2C, underlined sequence) (28). However, these three residues
are not conserved in the possum pIgR, suggesting that either the possum
receptor uses a different mechanism for basolateral targeting or that
the basolateral signaling sequence is larger than that previously
characterized. Interesting, amino acids immediately distal to these
residues are highly conserved between all the pIgR sequences (Fig. 2C, boxed), suggesting that they may form part of the
basolateral targeting signal.
The central region of the cytoplasmic domain, which has been implicated
to have a role in transcytosis, is the least conserved region between
all species (Fig. 2D) (26). The only amino acid shown to
have a direct role in transcytosis is conserved in the possum sequence
(Ser642; Fig. 2C), which lies outside of this
region (29). Phosphorylation of this serine in the rabbit pIgR
stimulates transcytosis in the absence of ligand binding. Truncation of
the carboxyl-terminal 30 aa has been shown to affect the endocytosis of
the receptor from the basolateral membrane (26). Part of this region is
highly conserved in the possum (Fig. 2E, boxed region) and
contains amino acid residues Ser703 and Tyr711
(of the possum sequence), which in addition to Tyr646 have
been shown in other species to be phosphorylated in response to ligand
binding, leading to endocytosis (30, 31).
Isolation of possum J chain gene
A 400-bp DNA fragment corresponding to the possum J chain was
amplified by RT-PCR using possum mammary gland total RNA (18 days
lactation) and oligonucleotide primers designed to conserved regions of
the J chain. This DNA fragment was used as a probe to isolate three
cDNA clones from the early lactation possum mammary gland cDNA library.
Two of these contained the entire J chain coding sequence, and the DNA
sequence was determined for the largest cDNA clone (1620 bp; Fig. 3). The possum J chain is 160 aa in
length and has a leader peptide of 22 aa. The possum J chain amino acid
sequence has 63–68% identity with eutherian sequences, which is
similar to that observed between eutherian species (67–80%). The
cysteine residues that form intra- and intermolecular disulfide bonds
are conserved (Fig. 3) (32).
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variable domain
The possum C variable and constant domains were amplified by 5'
RACE and RT-PCR, respectively, using total RNA from possum white blood
cells and oligonucleotide primers based on the known possum C
constant domain nucleotide sequence (17). The DNA sequence was
determined for two clones containing the variable domain, and a high
degree of amino acid variation was found in the
complementarity-determining regions (CDR1, CDR2, and CDR3-D-J) (V-1 and
V-2; Fig. 4). As expected, there were
also amino acid differences in the framework regions and in the leader
peptide sequence. The CDR3-D-J region is assembled by recombination of
the germline V (variable), D (diversity), and J (joining) segments
resulting in the differences in amino acid identity and length seen in
this region. The amino acid sequence of the constant domains (CH1–CH3)
of the New Zealand possum (C-2) differed from that obtained from the
Australian possum (C-1) at three amino acids: Asn144 to
Asp, Arg193 to Gly, and Asn399 to Lys (Fig. 4).
A recent study of the VH repertoire of the marsupial
Monodelphis domestica (South American short-tailed opossum)
demonstrated the presence of two VH families that are
phylogenetically most similar to the group III family in vertebrates
(33). The variable domains of the possum C are most homologous to
the MdoVH1 family (results not shown).
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Dimeric IgA is expressed by lymphocytes associated with the
mammary gland epithelium. The expression pattern IgA in the mammary
gland during lactation was determined by analysis of C and J chain
mRNA expression. Northern blots of total RNA extracted from mammary
glands at different stages of lactation were hybridized with the J
chain and IgA probes (Fig. 5A). The expression of both
these mRNAs was increased for two short periods during lactation: at
the beginning of lactation (days 1.5–6) and, unexpectedly, again in
the switch phase (days 106, 110, and 115). Expression of the J chain
mRNA during early lactation (days 31–94) was demonstrated using RT-PCR
(Fig. 5C). This suggests that there is expression of dimeric
IgA during this period of lactation, albeit at a low level.
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A). Importantly, we find that there are two periods during
lactation when expression of this mRNA was increased: at the beginning
of lactation (days 1.5–6) and again from the switch phase of lactation
until the end of lactation (days 106–189). It would appear
that the pattern pIgR mRNA expression coincides with that of the J
chain and C, with expression being highest at the beginning of
lactation and again from the latter part of the switch phase. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mRNAs of pIgR, C, and J chain were found to be expressed in the
mammary gland throughout lactation, suggesting that there is continuous
transfer of IgA into possum milk. Expression of C and J chain mRNAs
were dramatically increased at two specific periods of lactation, which
are summarized in Fig. 6. The first
period was at the beginning of lactation, and this would be consistent
with IgA transfer via the colostrum as seen in eutherians. The second
period of increased mRNA expression occurred for a short period (days
106–115) toward the end of the switch phase (80–120 days of
lactation). Expression of the pIgR mRNA was also increased at these two
stages of lactation, with the level of expression at the second period
remaining elevated to the end of lactation (Fig. 6). The second period
of increased expression of these mRNAs is likely to represent a second
period of enhanced IgA transfer via the milk. This second period
coincides with the end of the external gestation period of the pouch
young’s life and could be thought of as a second colostral phase.
Alternatively, the timing of this second period of IgA transfer, just
before pouch exit, suggests the intriguing possibility that this could
be equivalent to the placental Ig transfer that occurs during late
gestation in some eutherian mammals. Both periods of increased IgA
transfer would occur when the pouch young are exposed to new pathogens.
Indeed, there is a high rate of mortality in the possum pouch young at
birth and around the switch phase (34), and increased protection at
these stages would be advantageous.
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Colostral secretion of IgA is likely to be controlled by mammotrophic hormones affecting the expression of receptors and Igs. In mice administration of prolactin, progesterone and estrogen resulted in the development of the mammary epithelia with a concomitant increase in the number of associated IgA-secreting plasma cells and intraepithelia IgA (37). Interestingly, in marsupials including the possum there are two periods when serum concentration of prolactin is elevated, which coincide with increased dimeric IgA and pIgR expression in the mammary gland (38). There is a short peak before initiation of lactation and a second period of prolonged elevated serum prolactin concentration around the switch phase, increasing from approximately day 100 in the possum. An alternative possibility is that dimeric IgA expression is stimulated locally by the introduction of novel pathogens by the pouch young. The induction of an IgA Ab response by local challenge of the mammary gland is characteristic of eutherians and has been demonstrated in the quokka, Setonix brachyurus (35). However, this latter explanation would not explain the second period of increased pIgR mRNA expression.
In summary, we have cloned and characterized the pIgR and J chain of
the brushtail possum. The expression pattern of the J chain, C, and
pIgR mRNA during lactation suggests that there are two stages of
increased immune transfer in the possum at the beginning of lactation
and toward the end of the switch phase. We propose that these represent
two colostral phases during lactation, the second being unique to
marsupials.
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Acknowledgments |
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Footnotes |
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2 All sequences reported have been deposited into the GenBank database, accession nos. AF091138 (polymeric Ig receptor), AF091138 (J chain), and AF091139–AF091141 (IgA heavy chain).
3 Address correspondence and reprint requests to Dr. Frances M. Adamski, Dairy Science Group, AgResearch, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand.
4 Abbreviations used in this paper: pIgR, polymeric Ig receptor; IgA heavy chain, C; SC, secretory component; RACE, rapid amplification of cDNA end; CDR, complementarity-determining region.
Received for publication October 7, 1998. Accepted for publication February 26, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) from a marsupial—Trichosurus vulpecula (common brushtail possum). Immunol. Lett. 60:165.[Medline]
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