Analysis of the expression of immunoglobulins throughout lactation suggests two periods of immune...

Analysis of the expression of immunoglobulins throughout

lactation suggests two periods of immune transfer

in the tammar wallaby (Macropus eugenii)

Kerry A. Daly a,b, Matthew Digby b,c, Christophe Lefevre b,c, Sonia Mailer b,c,Peter Thomson a,b, Kevin Nicholas b,c, Peter Williamson a,b,*

a Centre for Advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science,

University of Sydney, NSW 2006, Australiab Cooperative Research Centre for Innovative Dairy Products, Australia

c Department of Zoology, University of Melbourne, Parkville, Vic. 3010, Australia

Received 12 March 2007; received in revised form 9 July 2007; accepted 17 July 2007

www.elsevier.com/locate/vetimm

Veterinary Immunology and Immunopathology 120 (2007) 187–200

Abstract

Marsupial young are born in an under-developed state without mature immune responses. Prior to the maturation of an immune

system, marsupial young are heavily reliant upon immune factors secreted in the milk to defend them against potential microbial

pathogens in the environment. In this study, we identified and characterized the immunoglobulin heavy chain constant regions, light

chains, polymeric Ig receptor (pIgR), J chain, neonatal Fc receptor (alpha chain) (FcRn) and the chemokine CCL28 from the model

marsupial species, the tammar wallaby (Macropus eugenii). Low levels of conservation were seen in motifs in Ca and Cg associated

with receptor binding and or transcytosis, and this may have potential implications for functionality. We evaluated the expression of

immunoglobulin genes in the tammar mammary gland throughout lactation and found that two periods of increased expression of

immunoglobulin genes occur. These two periods coincide with the birth of the young, and with its first emergence from the pouch.

This increased expression may represent a strategy for maternal immunological protection of the pouch young.

# 2007 Elsevier B.V. All rights reserved.

Keywords: Marsupial; Immunoglobulins; Maternal antibodies

Abbreviations: C, constant region; CDR, complementarity-deter-

mining region; EST, expressed sequence tag; FcRn, neonatal Fc

receptor; FR, framework region; Ig, immunoglobulin; ORF, open

reading frame; pIgR, polymeric immunoglobulin receptor; V, variable

region

* Corresponding author at: Centre for Advanced Technologies in

Animal Genetics and Reproduction, Faculty of Veterinary Science,

B19, University of Sydney, NSW 2006, Australia.

Tel.: +61 2 9351 3653; fax: +61 2 9351 2114.

E-mail address: [email protected] (P. Williamson).

0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetimm.2007.07.008

1. Introduction

Marsupial young are born in an altricial state of

development. Only the organ systems regarded as

essential for birth and perinatal survival are well

developed at parturition. The immune system, however,

is under-developed and naıve at birth. Pouch young do

not develop adult-like immune systems until just prior to

first leaving the pouch, at a period called the switch phase

(Baker et al., 2005; Basden et al., 1997; Belov et al.,

2002b; Block, 1960; Deane and Cooper, 1988; Old and

Deane, 2000). In the tammar wallaby (Macropus eugenii)

this is at 90 days of age (Basden et al., 1997) and in the

mailto:[email protected]

http://dx.doi.org/10.1016/j.vetimm.2007.07.008

K.A. Daly et al. / Veterinary Immunology and Immunopathology 120 (2007) 187–200188

brushtail possum (Trichosurus vulpecula), 70 days

(Belov et al., 2002b). In addition, the pouch itself

contains potentially pathogenic organisms at all stages of

the reproductive cycle (Deakin and Cooper, 2004; Old

and Deane, 1998). The secretion of antibodies and other

immune factors in the milk of the mother (Adamski and

Demmer, 1999, 2000; Adamski et al., 2000; Deane and

Cooper, 1988; Demmer et al., 1999), confers protection

to these immunologically naıve young during the early

post-partum period.

The marsupial lactation cycle differs from those seen

in eutherian species. Lactation is divided into four

phases. Phase 1 covers gestation and lasts for approxi-

mately 26.5 days in the tammar (Trott et al., 2003;

Tyndale-Biscoe and Renfree, 1987). During this phase,

all four glands will develop and produce milk (Hendry

et al., 1998; Trott et al., 2003). Phase 2A begins with the

birth of the young. The tammar neonate makes it way

from the cloaca to the pouch, where it will attach to one of

the four teats. The remaining teats will then regress.

During phase 2A, the pouch young is permanently

attached to this teat. Phase 2B or the switch phase begins

at 120 days in the tammar (Hendry et al., 1998; Trott

et al., 2003; Tyndale-Biscoe and Renfree, 1987). The

tammar neonate is still within the pouch, but begins to

suckle intermittently. Milk produced during phases 2A

and 2B is low in fat and protein, but high in carbohydrates

(Hendry et al., 1998; Trott et al., 2003). Phase 3 begins at

180 days and the tammar neonate begins to leave the

pouch (Trott et al., 2003; Tyndale-Biscoe and Renfree,

1987). The young begins to eat herbage and it will still

suckle, albeit less frequently and larger volumes. Milk

produced during this last stage is higher in volume,

protein and fat but lower in carbohydrates than milk

secreted in the earlier phases (Hendry et al., 1998; Trott

et al., 2003). This phase concludes with weaning and

involution at 300–350 days in the tammar (Trott et al.,

2003; Tyndale-Biscoe and Renfree, 1987).

The immunological significance of milk is a well-

studied area in eutherians. Immunoglobulins are usually

secreted in high amounts in the colostral milk of

eutherians. These immunoglobulins may be taken up by

the eutherian neonate’s gastrointestinal system and are

absorbed into their circulation in some species, thus

providing passive immunity. This absorption is limited

by the time taken for the gastrointestinal system’s

epithelium to close, and ranges from 24 h in some

ungulates, to up to 19 days in rodents (Kolb, 2002).

However, in marsupials absorption of immunoglobu-

lins, other proteins and possibly cells continues

throughout the entire lactation period (Cockson and

McNeice, 1980; Green and Renfree, 1982; Yadav, 1971;

Young et al., 1997). Metatherian immunoglobulin levels

in milk are much lower than those seen in eutherians

(Basden et al., 1997; Deane et al., 1990). Consequently,

marsupials must rely upon more efficient uptake and

different immunoglobulin isotypes to help meet the

immunological challenges of the young (Deane et al.,

1990). The brushtail possum has two periods of immune

transfer during lactation and these periods coincide with

the periods of greatest immune challenge for the pouch

young—i.e. birth and leaving the pouch (Adamski and

Demmer, 1999, 2000; Adamski et al., 2000; Demmer

et al., 1999). These two phases of immune transfer in the

mammary gland of the brushtail possum has been

demonstrated for not only immunoglobulins (Adamski

and Demmer, 1999, 2000) but also other immune

components such as the neonatal Fc alpha receptor

(FcRn) (Adamski et al., 2000) and the iron regulatory

proteins ferritin and transferrin (Demmer et al., 1999).

This pattern of differential expression occurs in the

gastrointestinal tract of the brushtail possum young

late in lactation (Western et al., 2003). Consequently,

differential regulation of expression of immune

components in both the mammary gland of the mother

and the gastrointestinal tract of the young may help the

marsupial neonate to survive periods of immune stress

while still relatively immunocompromised.

In this study, we have identified and examined the

expression of immunoglobulin heavy and light chains,

the J chain, the polymeric immunoglobulin receptor

(pIgR), the neonatal Fc receptor (FcRn) and the IgA

secreting cell specific chemokine CCL28 in the

mammary gland of the tammar wallaby. This work

shows that the tammar wallaby possesses all heavy and

light chain isotypes previously identified in marsupials

(IgA, IgE, IgG, IgM, Igk and Igl). In addition, we have

examined the functional regions of these immunoglo-

bulins and their receptors. While there was a high level

of conservation present in many functional motifs of

immunoglobulins and their receptors, there were some

non-conservative substitutions in a motif important in

pIgR binding in IgA that may affect polymerisation and

transcytosis of this mucosal immunoglobulin (Aves-

kogh and Hellman, 1998; Aveskogh et al., 1999; Belov

et al., 1998, 1999a,b,c, 2001, 2002a; Lucero et al., 1998;

Miller et al., 1999), and in an FcRn-binding affinity

motifs in IgG. Two distinct periods of increased

immune expression were observed for most of these

immune components in the tammar mammary gland.

This suggests that differential regulation of immuno-

globulins and their receptors is a generic marsupial

mechanism and may be an important defence strategy

for the survival of pouch young.

K.A. Daly et al. / Veterinary Immunology and Immunopathology 120 (2007) 187–200 189

2. Materials and methods

2.1. Animals

Tissue was obtained from a colony of tammar

wallabies (M. eugenii) kept in open grassy yards at the

Victorian Institute of Animal Science, Attword,

Victoria, Australia. Food and water was provided ad

libertum. All procedures were carried out according the

guidelines of the experimentation ethics of the Victorian

Institute of Animal Science.

2.2. Expressed sequence tags (EST) library

construction

Twenty cDNA libraries were prepared using tammar

wallaby mammary gland RNA from day 23 of pregnancy

(n = 4), from lactating glands at day 130 (n = 4), day 260

(n = 1), day 130 subtracted for all the major milk protein

genes (n = 2), non-lactating (n = 2) and a normalized

library of the combined RNA from pregnant animals at

day 26, lactating at days 55, 87, 130, 180, 220, 260 and

involuting tissue at day 5 (Lefevre et al., submitted for

publication). The cDNAs were directionally cloned, and

sequenced from the 5-prime end of the insert. A total of

15,001 expressed sequence tags that were greater than

100 bases in length and had less that 5% ambiguity were

obtained. Assembly into contigs was carried out using a

PhredPhrap (Ewing and Green, 1998; Ewing et al., 1998;

Gordon et al., 1998). Sequences were compared to

GenBank, Unigene and SwissProt, using the basic local

alignment search tool (BLAST) algorithm for annotation.

An E value of 1E�8 was taken as the cut off value for a

significant alignment.

2.3. Identification of heavy chain constant regions,

light chains and immunoglobulin receptors from

tammar mammary gland EST library

Immunoglobulin clones were isolated from the

database using immunology related gene ontology

numbers. The EnsMart package (release version 18.1)

was used for screening the database. Comparison with

known marsupial and eutherian sequences was used to

identify domains and conserved features.

2.4. Alignment and comparison of sequences

All other alignments were done on the amino acid

sequences. Nucleotide sequences were translated

using the translate tool from the ExPASy site (http://

au.expasy.org/tools/dna.html). Pairwise alignments

were carried out using the SSEARCH program (version

3.0) (Smith and Waterman, 1981) from the Georgetown

Pairwise Alignment site (http://pir.georgetown.edu/

pirwww/search/pairwise.html). Identity percentages,

E values, Smith–Waterman scores and the total length

of alignment were all evaluated when assessing

homology between protein sequences. E values of less

than 1E�8 was taken as the cut off value for a significant

alignment. All multiple alignments used the CLUS-

TALW (Thompson et al., 1994) program available

through BioManager by ANGIS (Australian National

Genomic Information Service, NSW, Australia)

(www.angis.or.au). Multiple alignments were prepared

for publication using BOXSHADE 3.21 program

(Hoffman, K and Baron MB) (http://www.ch.embne-

t.org/software/BOX_form.html). Determination of

areas of functional significance was done by compar-

ison with previously characterized sequences on

multiple alignments. Sequences used in alignments

and analyses are listed in Table 1.

2.5. RNA preparation and analysis of gene

expression by microarray

Custom made cDNA microarrays were designed

based on the tammar mammary gland EST library. Each

slide was printed with 10,000 ESTs from this cDNA

library. Tissue samples from the mammary glands of

virgin (three samples), pregnant (days 5, 22 and 25)

and lactating (days 26, 30, 105, 155, 193, 205, 238, 245

and 285) were collected for microarray analysis. In

addition, involuting mammary tissue generated by

forced weaning at 290 days was collected from tammars

at 291, 295 and 300 days (i.e. 1, 5 and 10 of involution).

Total RNA was isolated from these tissue samples using

QIAGEN RNeasy mini kits (Sydney Australia) and

following the manufacturer’s instructions. Thirty-six

comparisons were made using 72 microarray slides,

which covered the entire lactation cycle, allowing a

complete analysis of pregnancy, lactation and involu-

tion. Dye swapping was also used to prevent the

influence of dye-based bias in the results.

RNA from each treatment group was labeled using

amino allyl reverse transcription followed by Cy3 and

Cy5 coupling. Samples of total RNA (50 mg) were

reverse transcribed using oligo dT (87 ng/ml) MMLV

reverse transcriptase (Promega), RNAse H and 1� buffer

at 42 8C for 2.5 h. The reaction mix was hydrolyzed by

incubation at 65 8C for 15 minutes in the presence of

33 mM NaOH, 33 mM EDTA and 40 mM acetic acid.

The cDNAwas then adsorbed to a Qiagen QIAquick PCR

Purification column. Coupling of either Cy3 or Cy5 dye

http://au.expasy.org/tools/dna.html

http://au.expasy.org/tools/dna.html

http://pir.georgetown.edu/pirwww/search/pairwise.html

http://pir.georgetown.edu/pirwww/search/pairwise.html

http://www.angis.or.au/

http://www.ch.embnet.org/software/BOX_form.html

http://www.ch.embnet.org/software/BOX_form.html


Table 1

Sequences used in analysis

Ca Brushtail possum (Trichosurus vupecula) AAD41690, grey short-tailed opossum (Monodelphis domestica) AAC48835, mouse

(Mus musculus) AAB59662, echidna (Tachyglossus aculeatus), platypus (Ornithorhynchus anatinus) A1 AAL17700,

platypus A2 AAL17701, human (Homo sapiens) A2HU, cow (Bos taurus) I45927, rabbit (Oryctolagus cuniculus) P01879,

chicken (Gallus gallus) A46507, rat (Rattus norvegicus) AAA41373 and chimpanzee (Pan troglodytes) CAA30841

Ce Brushtail possum AAF80357, grey short-tailed opossum AAC7964, cow AA037095, dog (Canis familaris) AAD24458,

horse (Equus callabus) AAA85662, mouse CAA25977, chimpanzee AAA35416, human AAB59424, sheep AAA51378,

pig AAC48776, platypus AAL17702 and echidna AAM45140

Cg Brushtail possum AAG28392, grey short-tailed opossum AAC79675, human1 P01857, human2 P01859, human4 P01861,

mouse P01863, rabbit P01870, sheep S31459, cow S06611, rat NP_001014103, pig AAD38418, chimpanzee XP_522970,

platypus G1 AAL17703, platypus G2 AAL17704 and echidna AAM61760

Cm Brushtail possum AAF80358, grey short-tailed opossum AAC21191, cow AF005274, human X67301, chicken X01613,

mouse X03690, platypus U27213, echidna AAN33013, rabbit P03988, sheep (Ovis aries) X59994, pig (Sus scrofa)

AAA51297, rainbow trout (Oncorhynchus mykiss) AAW66975, haddock (Melanogrammus aeglefinus) CAF22022, bastard

halibut (Paralichthys olivaceus) BAC99314, bowfin (Amia calva) AAC59687 and longnose gar (Lepisosteus osseus) AAC59688

Igk Brushtail possum AAL17619, grey short-tailed opossum AAF2554, echidna AAO84653, platypus AAO84650, dog P01618,

human BAA14189, mouse CAA36033, pig PT0219, rabbit AAR26308, rat AAB21182, African clawed frog S14077 and

nurseshark AAA50248

Igl Brushtail possum AAL37214, grey short-tailed opossum AAC98626, platypus AAO16039, mink (Mustela vison) CAA39711,

horse S17598, human CAA32725, mouse AAC52488, pig PT0220, rabbit BAA20955, rat C27390, sheep B30554,

chicken P20763 and sandbar shark (Carcharhinus plumbeus) I51383

pIgR Tammar AAK69596, brushtail possum AAD41688, human AAQ88909, cattle NP_776568, mouse NP_035212, chicken

AAQ14493, pig NP_999324

J chain Brushtail possum AAD41689, mouse NP_690052, earthworm (Lumbricina subclass) AAC12908, human NP_65324,

chicken BAB83927, rabbit P23108, dog AAL91654, cattle NP_78967, red-eared slider turtle (Trachemys scripta) BAC22087,

nurseshark (Ginglymostoma cirratum) AAO14897, bullfrog (Rana catesbeiana) AAF25773 and African clawed frog

(Xenopus laevis) AAC05636

FcRn Brushtail possum AAG28391, mouse NP_034319, human AF200220, pig NP_999362, sheep AAN31410, crab-eating macaque

(Macaca fascicularis) AAL92101, cattle AAF60957 and Arabian camel (Camelus dromedaries) AAX82484

CCL28 Human NP_683513, pig NP_001019866, macaque (Macacca mulatta) NP_0010280419, dog NP_001005257, rat NP_446152

and mouse NP_064675

was performed by incubation with adsorbed cDNA in

0.1 M sodium bicarbonate for 1 h at room temperature in

darkness, followed by elution in 80 ml water. Labeled

cDNA was further purified using a second Qiagen

QIAquick PCR Purification column. Cy3 and Cy5 labeled

probes in a final concentration of 400 mg/ml yeast tRNA,

1 mg/ml human Cot 1 DNA, 200 mg/ml polydT50, 1.2�Denharts, 1 mg/ml herring sperm DNA, 3.2� SSC, 50%

formamide and 0.1% SDS were heated to 100 8C for 3

minutes. Probes were hybridised overnight at 42 8C in a

humidified chamber and transferred onto the custom

tammar wallaby EST microarray. Microarrays were

washed in 0.5� SSC, 0.01% SDS for 1 minute, 0.5�SSC for 3 minutes then 0.006� SSC for 3 minutes at

room temperature in the dark. Microarray slides were

scanned with an Agilent Scanner and images analyzed

using Versarray Software (Biorad). Loess normalization

was performed to normalize the data within and between

the arrays (Yang et al., 2002). All the normalized

expression level data were then analyzed simultaneously

using a large-scale linear mixed model. This model

included random effect terms to describe both the

physical design of the microarrays, as well as the gene

effects and gene-contrast effects (Thomson, 2006). The

BLUPs (best liner unbiased predictor) of the gene effects

are then fitted to a mixture model of the form

p1Nð0; s2g þ s2

eÞ þ ð1� p1ÞNð0; s2eÞ, where p1 is the

(prior) probability of a gene being DE, s2g the variance of

the underlying expression levels of the DE genes, and s2e

is the residual variance, common to DE and non-DE

genes. The mixture model is fitted using the EM

(expectation maximisation) algorithm, and in the

process, returns the probability of the gene not being

differentially expressed ( pde) and that the effect of the

gene was not different from that in the previous time point

in lactation ( pdiff).

3. Results

3.1. Identification and characterization of

sequences

From the tammar EST library, partial sequences of

Cm, Ca, Cg, Ce, FcRn, J chain, pIgR and full length

sequences of Igk and Igl were isolated. Isolated tammar

Cm, Ca, Cg and Ce sequences were compared with


known marsupial sequences from the brushtail possum

and the grey short-tailed opossum. The tammar sequence

for Cg (EF599608) was deduced from a single clone and

was 740 nucleotides in length, which translated to an

open reading frame (ORF) of 246 residues in length.

When this was compared to the grey short-tailed

opossum, the tammar Cg was composed of the hinge

region and domains two and three (Supplementary data,

Fig. 1). Tammar Cg had a protein percentage identity of

78% when compared to the opossum and 57% to

eutherians. The tammar Cm sequence was 1377

nucleotides in length, which translated to an ORF of

270 amino acids. Sequences from two clones (EF599609

and EF599610), overlapping by 188 identical residues,

which were also identical at the nucleotide level, were

used to determine the Cm sequence. When compared

with known marsupial Cm, this tammar Cm was

composed of domains two to four and had a peptide

percentage identity of 82% (Supplementary data, Fig. 2).

This protein identity decreased to 63% with eutherians.

The tammar Ca (EF599606) was formed from one clone

and was found to be 1310 bases in size. This gave an ORF

of 191 residues when translated and was composed of

domains two and three when compared to known

metatherian Ca (Supplementary data, Fig. 3). Tammar

Ca demonstrated a peptide percentage identity of 80% to

other marsupials and 55% to eutherians. Tammar Ce was

the smallest gene isolated, at 737 nucleotides, which

came from a single clone (EF599607). When compared

to known metatherian sequences (Supplementary data,

Fig. 4), the tammar Ce peptide contained the second to

fourth domains with a protein identity of 79%.

Two light chains were also identified from the

library. Igk and Igl were each formed from single full

length clones. Igk was 744 nucleotides in length and

translated to give a protein of 240 residues (EF599616).

When compared with the previously identified brushtail

possum (Belov et al., 2001) and grey short-tailed

opossum sequences (Miller et al., 1999), the variable,

constant and J segments were all present (Supplemen-

tary data, Fig. 5). The amino percentage identity for the

entire Igk was 78% with other marsupials and 61% with

eutherians. The constant region (Ck) (from residues 136

to 240) showed high levels of identity with an average

peptide percentage identity of 83% with other marsupial

sequences. The variable region (Vk) had an average

protein percentage identity of 69% when compared to

other known marsupial sequences. However three other

Igk clones that contained full length Vk regions were

also identified (EF599617, EF599618 and EF599619).

The average amino percentage identity for the Vk

region for these clones compared to the full length

tammar Igk sequence was 68%. There were high levels

of homology in the framework regions (FR) when these

tammar Vk clones were compared to other mammalian

sequences (Supplementary data, Fig. 5). This included

in FR2, which has been associated with antigen-binding

affinity and heavy chain binding (Masuda et al., 2006).

In the full length tammar Igk clone (EF599616) a five-

residue insertion was seen in CDR1 and this was shared

with the brushtail possum, opossum and mouse. This

insertion was well conserved, with a serine and glycine

common in all.

Igl was 750 nucleotides in length and this gave a

250-residue peptide (EF599620). Comparison with

previously identified brushtail possum and opossum

sequences (Lucero et al., 1998) revealed that the

isolated tammar sequence again included the variable, J

and constant segment (Supplementary data, Fig. 6).

Amino percentage identities with other metatherian Igl

was 77% and 59% with eutherians. The Cl spans

from residues 139 to 187 and has an average percentage

identity with other marsupial peptides of 77%. The

Vl region had an average protein identity with

other known marsupial sequences of 60%. There were

ten other clones identified that had complete Vl

regions (EF599615, EF599621, EF599622, EF599623,

EF599624, EF599625, EF599626, EF599627, EF-

599628 and EF599629), with an average amino

percentage identity of 62% compared to the full length

tammar Igl clone. While again the FR regions had high

levels of conservation, variation and little homology

was seen in CDR1 and CDR3 amongst the tammar Vl

clones (Supplementary data, Fig. 6). Homology

between constant regions of Igl and Igk were much

lower with an average 40% identity seen with other

marsupial peptide sequences.

Also identified in the database were the sequences for

the pIgR, FcRn, J chain and the chemokine CCL28.

Several tammar pIgR clones were identified in the library.

These clones were clustered around the carboxyl

terminal of tammar pIgR (AAF69593) and had an

average protein percentage identity in this overlapping

region of 100%. The FcRn sequence was 1304

nucleotides, which gave an ORF of 375 amino acids.

The tammar FcRn protein sequence had a percentage

identity of 89% with other marsupials and 51% with

eutherians. Tammar FcRn was composed of two clones

(EF599613, EF599614), one that formed residues 1–232,

and the other from 233 to 375, with a percentage identity

of 89% in the overlapping area. The J chain sequence was

also isolated from the database (EF599612). The J chain

sequence was 728 nucleotides in length but translated to

give an ORF of 93 residues. These residues formed the


carboxyl terminus of the peptide and the protein lacked

83 amino acids from the amino terminus. This sequence

was derived from a single clone and has a protein

percentage identity of 83% with the previously identified

sequence in the brushtail possum (Adamski and Demmer,

1999). CCL28 was also examined in this study due to its

role in chemotaxis of IgA secreting-plasma cells to the

mammary gland. CCL28 was also derived from one

clone (EF599611) and was 713 nucleotides long, which

translated to give an ORF of 109 residues. High

percentage identities were seen on comparison of this

chemokine with homologous proteins (an average

identity of 63% with eutherians), however low levels

of conservation are generally expected in chemokines

and cytokines due to high rates of evolution (Harrison and

Wedlock, 2000).

3.2. Conservation of functional and structural

features

The level of homology in the constant regions of the

immunoglobulin subtypes was high. There was good

conservation of cysteine and tryptophan residues in all

four heavy chain constant regions. The tammar Cg

sequence shares two out of the four previously identified

potential N-linked glycosylation sites of the brushtail

possum (Belov et al., 1999a) (Supplementary data, Fig.

1). Variable levels of conservation of FcRn affinity and

binding motifs in the Cg2 and Cg3 domains were also

found (Table 2). Little conservation of the 252–256

motif, which has been associated with binding affinity

for the neonatal Fc receptor (FcRn) (Dall’Acqua et al.,

Table 2

Motifs involved in FcRn transcytosis in Cg2 and Cg3 domains

Species 252–256 310–311 433–436

Tammar TLSRV HQ PNQN

Brushtail TLSRV HQ PNQT

Opossum KLSRS HQ PNQI

Platypus SVAGT SK PQKF

Echidna SVTGT SK PQKF

HumanG1 MISRT HQ HNHY



Rabbit MISRT HQ HNHY

Sheep TISGT HQ HNHY

Cow MITGT HQ HNHY

Mouse MISLS HQ HNHH

Rat TITLT HR HNHH

Pig MISQT HQ HNHY

Polymorphism in these three areas has been linked to variations in the

binding affinity of IgG for FcRn and consequent variations in the

transcytosis and serum persistence of IgG in eutherians. Details of the

sequences used are listed in Section 2.4.

2006; Medesan et al., 1997), was found between

eutherians and lower order mammals (Table 2). Only

Ser254 was preserved in marsupials and the majority of

eutherians. His310 and Gln311, which are also involved

in FcRn binding (Medesan et al., 1997), are present in

all therian mammals but absent in monotremes, where

there is a non-conservative substitution of Ser310 and

Lys311. Poor preservation of the 433–436 motif was also

seen between eutherians and lower order mammals

(Table 2). This area has been associated with the

regulation of transcytosis and serum persistence of IgG

(Medesan et al., 1997).

Tammar Ce demonstrates higher levels of conserva-

tion with four potential N-linked glycosylation sites seen

in the brushtail possum (Belov et al., 1999b), including

the two that are specific for marsupials (amino acids 30

and 225) (Supplementary data, Fig. 4). All four sites align

well with the domains already identified in grey short-

tailed opossum and brushtail possum peptides. The Cm4

domain in all marsupial species contains a terminal

cysteine residue and glycosylation site (Supplementary

data, Fig. 2). These features and the 18-residue tail piece

that surrounds them are highly conserved in vertebrates

(de Lalla et al., 1998). Residues Cys337 in the Cm2 and

Cys414 in the Cm3 domains, which are involved in

functional disulphide bonds (Braathen et al., 2002), are

both conserved in all species examined, including the

tammar (Supplementary data, Fig. 2).

Tammar Ca also had a variable level of preservation

of functional motifs, such as those involved in

interactions with the J chain and pIgR. The 18-residue

tail piece (box 3 in Fig. 1) had very high levels of

conservation, with an average percentage identity of

78%. The penultimate cysteine was present in all

species examined. Motif 440–443 (box 2 in Fig. 1),

which has been shown to be required for optimal

interaction with human pIgR (Hexham et al., 1999), is

extremely well conserved. However in all marsupials

and monotremes there is an Asn442 rather than an

alanine. Motif 401–414 shows very little conservation

(box 1 in Fig. 1). This motif has been associated with

pIgR binding in humans, especially the latter part of the

motif which forms a loop (Hexham et al., 1999). This

loop is associated with a three threonine insertion in this

motif, when the area is compared to the equivalent area

in Cg4 (Hexham et al., 1999). This latter part of the

motif is better conserved, however the platypus has a

Ser408, Ala409 and Tyr414, the Echidna a Ser408, Pro409

and Tyr414, and the brushtail possum and tammar have

an Asn409 and Ile411 (box 1 in Fig. 1). The remaining

polymorphisms in this loop area are conservative. There

are also some residues within the Ca2 domain that are


Fig. 1. Alignment of the a3 domain of the heavy chain constant area of IgA. Boxes indicate areas of importance in interaction with pIgR. Numbered

boxed are 1, 401 motif; 2, 440 motif; 3, the a tail piece. Poor conservation is seen in the first motif in lower order mammals.

important for IgA interactions, such as Cys311 and

Cys377 (Braathen et al., 2002; Sorensen et al., 1999),

which were found to be conserved in all sequences

examined.

Interactions between the heavy and light chain

components of the immunoglobulin molecule occur in

both the constant and variable chains. Table 3 lists the

contact residues for Cl and Ck regions based on those

previously identified (Miller, 1991). These were not

determined for the heavy chains as tammar sequences

were incomplete. Several residues, but not, all showed a

high level of conservation. A phenylalanine at residue

120 was conserved in almost all species examined, as

were serines at positions 175 and 177. Conservative

substitutions were generally seen at 133, 139, 164

(serines or threonines) and 179 (aromatic residues).

There was a high level of conservation of motifs

involved in IgG binding and basolateral transport in

FcRn (Fig. 2). Residues 74–91 (box 1 in Fig. 2) form an

alpha helix that is thought to act as a modulator of FcRn

Table 3

Residues involved in CL–CH interactions in Igl and Igk

118 120 133 138 139 1

Opossum l M Y T I S L

Tammar l N F T L N N

Brushtail l N F T L S D

Human l T F T I S E

Mouse l S F T L N L

Tammar k F F S V N L

Brushtail k F F S V N V

Human k F F S L N Q

Mouse k S F S L N L

Conservation of residues was seen for residues 120, 133 139, 164, 175, 17

key interactions due to its spatial proximity to other

functionally relevant residues (Zhou et al., 2005). There

are high levels of conservation of the residues within

this motif. Based on the tertiary structure, residue 137

(indicated by an asterisk in box 2, Fig. 2) is spatially

close to this first motif. Residue 137 and the motif

around it (box 2 in Fig. 2) forms an alpha helix that also

has been shown to have a role in IgG binding (Zhou

et al., 2003, 2005). Only Trp143 is well conserved within

this second motif, suggesting a common structural or

functional role for it. Two other functionally important

motifs have been identified in the carboxyl terminus of

FcRn and these have been shown to be important in the

basolateral transport of FcRn (Newton et al., 2005)

(boxes 3 and 4 in Fig. 2). There are very high levels of

conservation of the YXXF (box 3 in Fig. 2) and

DDXXXLL motifs (box 4 in Fig. 2) between all species

examined. Ala313, Trp315 and Lys318 have all been

shown to be important in the YXXF motif and the first

two were found in all sequences. Lys318 was much less

62 164 167 175 176 177 179

S V S A S Y

A S S T S F

T S S A S Y

T S A A S Y

S D S M S T

S D S L S T

S E S L S T

S E S L S T

S D S M S T

7 and 179. Details of the sequences are listed in Section 2.4.


Fig. 2. Alignment of FcRn Sequences. Numbered boxed areas refer to motif involved in binding of IgG by FcRn or the basolateral transport of

FcRn. 1, Residues 74–91 of the a1 helix of FcRn; 2, 136–147 in the a2 helix of FcRn; 3, YXXF motif; 4, DDXXXLL motif. The asterisk marks

residue 137.

conserved with a phenylalanine present in sheep, cattle,

camel and pig, and an arginine in both marsupial

species.

There was conservation of the cysteine residues in

the J chain of the tammar. Eight cysteine residues have

been identified in eutherians, five of which were present

in the tammar sequence. Two of these cysteines were in

the missing amino terminus, but were present in the

brushtail possum. In addition, there was a non-

conservative substitution of Cys70 for a serine in the

tammar sequence. Conservation of functional features

for tammar pIgR has been previously described (Taylor

et al., 2002). High levels of homology and conservation

are found in all mammalian sequences in domain 1 and

CDR2, which have been demonstrated to be important

in Cm4 and Ca3 interactions (Johansen et al., 2000;

Kaetzel, 2005).

The tammar CCL28 putative protein, although

incomplete at the carboxyl terminus, still demonstrated

many of the structural features of this class of

chemokine. Chemokines generally have four cysteines,

which form two disulphide bonds and upon which

classification into families is based. However, CCL28

differs slightly, in that it has six cysteines (Wang et al.,

2000) that may have a functional role in stabilisation of

the core of the chemokine (Zabel et al., 2006). In

comparison, tammar CCL28 has five of these six

cysteines. These cysteines included the characteristic

CC at 31–32, for which CCL28 is classified as a CC

chemokine. A potential N-linked glycosylation site

present in the human, mouse, rat and macaque proteins

is also preserved in the tammar sequence.

3.3. Expression in the lactating mammary gland

The immunoglobulin isotypes displayed a spike of

high expression between 25 and 26 days of the lactation

cycle (that is around the birth of the pouch young at 26.5

days) (Fig. 3A and B). In the case of IgM and IgE, this

peak at days 25–26 was significant ( pde < 0.05). In all

immunoglobulin isotypes there is another high expres-

sion effect in the later phases of lactation (late 2B and 3)

(Fig. 3A). In the case of IgM, this late positive effect in

expression begins around day 193 (middle of the switch

phase) ( pdiff < 0.05). IgG expression also demonstrated

significant low expression effects early in the switch

phase, then increased late in this phase and phase 3 of

lactation and in involution. CCL28 expression effect

(Fig. 3A) was significantly down-regulated in samples

from virgin animals ( pde < 0.01), then demonstrated a

peak positive effect in mRNA expression around

parturition (days 26–30, pde < 0.001). High CCL28

expression was also observed in the switch phase, at the

end of phase 3 and during early involution.


Fig. 3. Expression of immunoglobulins, receptors and CCL28 in the lactating mammary gland of the tammar wallaby. (A) Expression of

immunoglobulins and CCL28, (B) expression of immunoglobulin light chains and (C) expression in immunoglobulin receptors. The days of lactation

are on the X-axis and the genes on the Y-axis. The phases of lactation are indicated on each graphs. v, virgin; b, indicates birth of the young; I,

involution; 1, 2A, 2B and 3 indicate the phases of lactation.

Expression of immunoglobulin light chains and

immunoglobulin receptors followed a similar pattern

(Fig. 3B and C). All had a small up-regulation in effect

around parturition (days 26–30). The expression effect of

the immunoglobulin light chains (the k and l chains) was

high from day 193 of lactation (Fig. 3B). This is highly

significant for Igl ( pde < 0.001) and continued into

involuting mammary gland (days 291–300). Prior to this,

Igl had a significantly low expression in virgin animals

and from days 30 to 155. pIgR and the J chain

demonstrated similar patterns of expression (Fig. 3C).

Both had a significant ( pde < 0.001) down-regulation in

expression in virgin and low levels of expression in

pregnant animals. Both also had a subtle increase in

expression around parturition (days 26–30) but a more

significant up-regulated effect occurred from late in

phase 2A and continued throughout the cycle and into

involution ( pde range from <0.001 to <0.05). The

neonatal FcRn illustrated a very different expression

pattern throughout lactation when compared to the other

immunoglobulin molecules and their receptors (Fig. 3C).

FcRn still showed a sharp positive peak in expression at

day 26 (that is around parturition). However FcRn had a

small up-regulation in effect in virgin animals, during the

switch phase and in the involuting gland. The increased

expression seen in expression in the majority of other

immunoglobulin related genes in the later phases of

lactation was not apparent in FcRn.

4. Discussion

Milk has roles in not only nutrition, but also immune

defences that are crucial for neonatal survival. In this

study we have demonstrated differential regulation of

three of these immunoglobulin heavy chain isotypes,

corresponding light chains, receptors (pIgR, FcRn and J

chain) and an associated chemokine (CCL28) in a

macropod marsupial, the tammar wallaby, across the

lactation cycle. These results suggest that differential

regulation of immunoglobulins throughout the lactation

cycle in marsupials may be an important immune defence

strategy for the mother’s mammary gland and the pouch

young, as these periods of increased immune transfer

occur at times of increased immune challenge (i.e. birth

and leaving the pouch). In addition, we have analyzed

functionally important regions of these immunoglobulins

and their receptors. While the majority of motifs appear

to have high levels of homology, lower levels of

conservation were seen in a pIgR binding motif in

tammar Ca, which may have implications for IgA and

mucosal immunity, and motifs in Cg that may affect

FcRn affinity, transcytosis and serum IgG persistence.


Marsupial young are born in an altricial state, with

only those systems that are required for perinatal

survival well developed. Mature immune responses do

not develop in marsupial young until they are about to

leave the pouch, in a period known as the switch phase

(Baker et al., 2005; Basden et al., 1997; Belov et al.,

2002b; Deane and Cooper, 1988; Old and Deane, 2000).

Prior to this, some immunological protection may be

provided by the gradual maturation of immune organs,

such as the cervical thymus, which is the first immune

organ to develop at 3 weeks in the tammar (Old and

Deane, 2000). However, prior to immune maturity,

protection is provided predominantly by the secretions

in the milk (Adamski and Demmer, 1999, 2000;

Demmer et al., 1999) and possibly from the pouch skin

(Bobek and Deane, 2002; Deakin and Cooper, 2004;

Old and Deane, 1998). Milk containing immunoglo-

bulins makes a large contribution to the protection of the

marsupial young as these immunoglobulins are able to

be taken up throughout the entire lactation cycle

(Cockson and McNeice, 1980; Green and Renfree,

1982; Yadav, 1971; Young et al., 1997).

This study identified and characterized immunoglo-

bulin isotypes from a mammary gland cDNA library.

Immunoglobulin isotypes such as IgG, IgM, IgE, Igk and

Igl were also found to have similar patterns of expression

throughout lactation in the tammar wallaby. IgE has not

been detected in the milk or mammary gland of any

marsupial species before this study. Instead IgA (and not

IgG) is the predominant immunoglobulin isotype in the

milk of the tammar (Deane et al., 1990) and brushtail

possum (Adamski and Demmer, 1999, 2000). In this

study the chemokine CCL28, which plays a key role in

enabling the passive transfer of IgA from the mother to

infant in eutherians (Meurens et al., 2006; Wilson and

Butcher, 2004), has similar expression patterns as the

immunoglobulin isotypes identified in this study and

previous studies of IgA expression in marsupial milk

(Adamski and Demmer, 1999, 2000). Differential

regulation of CCL28 demonstrated its importance around

parturition, the switch phase and involution. Increases in

its expression may reflect increased periods of IgA

transfer in the tammar, such as immediately post-partum.

IgA plays an important role in mucosal immunity and so

would provide local protection in the gut of the neonate

rather than being absorbed systemically. The tammar

CCL28 protein demonstrated a high degree of homology

to eutherian counterparts, but was missing one of the

additional cysteines present in eutherian CCL28. It is

unknown if this missing cysteine would have an effect on

the stability of the core of the protein and hence the

function of CCL28.

This is also the first study to examine the expression

of IgM in the mammary gland of the tammar, although it

has been found in the milk of the tammar previously.

The differential pattern of expression of IgM throughout

lactation seen in this study has not been previously

reported for this immunoglobulin subtype. Both IgA

and polymeric IgM have important roles in mucosal

immunity. Microbial colonization of the pouch young

begins immediately post-partum in the non-sterile

environment of the mother’s pouch. The gut flora of

marsupials has been shown to alter dramatically during

phase 3 (Yadav et al., 1972) as they begin to eat herbage

and become exposed to new micro-organisms. This

concurrent differential expression of mucosal immu-

noglobulins during lactation in both the tammar and the

brushtail possum (Adamski and Demmer, 1999, 2000)

suggests the importance that mucosal immunity may

have in marsupial neonatal survival, development of

mucosal microflora in the pouch young and mammary

gland immunobiology.

This differential pattern of expression was also seen

in the mucosal immunoglobulin associated receptors—

the polymeric immunoglobulin receptor (pIgR) and the

J chain. Both of these receptors are involved in the

transcytosis, polymerisation and also protection of these

mucosal immunoglobulins in the gastrointestinal

system (via the secretory component, which is formed

by cleavage of pIgR). Both pIgR and the J chain had

high expression at parturition, late in the switch phase,

and continued through into involution. This again

concurs with the expression of the mucosal immuno-

globulins. This may be a reflection of the excretion of

these immunoglobulins by the mammary gland and in

particular of the dominance of IgA in the milk of the

tammar. pIgR is cleaved to form the secretory

component (SC), which has its own protective roles

against bacterial adherence (Hughes et al., 1997;

Kaetzel, 2001). Consequently increased expression of

pIgR may also reflect the immune contribution of the

SC into mammary gland secretions.

In general, there were high levels of conservation of

those motifs involved in pIgR and J chain functionality

in Cm and Ca. There is a high degree of homology and

conservation of domains and functional features in pIgR

of the tammar (Taylor et al., 2002), including domain 1

and the CDR2 that have been identified as interacting

with the Cm3 and Ca4 chains (Johansen et al., 2000;

Kaetzel, 2005; Lewis et al., 2005; Yoo et al., 1999). The

C-terminal tail of both Cm and Ca showed very high

levels of homology between species. In all marsupial

species examined, there was conservation of the

penultimate tail piece cysteine, several other disulphide


structural bond cysteines in Cm and Ca, and a N-linked

potential glycosylation site in Cm, which is important in

the binding of the J chain, cellular transport and

consequently polymerization (Johansen et al., 2000;

Lewis et al., 2005; Sorensen et al., 2000). However, in a

motif identified as playing a key role in pIgR binding of

Ca3 in humans (Hexham et al., 1999; Lewis et al.,

2005), marsupial and monotreme sequences had low

levels of homology. The latter part of this motif forms a

loop, which interacts with pIgR (Hexham et al., 1999;

Johansen et al., 2000). The tammar, brushtail possum

and both monotreme Ca sequences all have non-

conservative substitutions in these latter loop residues.

This area is critical for Ca–pIgR interaction (Hexham

et al., 1999; Lewis et al., 2005) and the variation in this

motif may have implications for transcytosis and

polymerization of IgA in these lower order mammals.

The protein levels of IgA and IgM previously reported

in the milk of the tammar (Deane et al., 1990) suggests

this motif is not essential for expression, but the effects

may also be felt in the interactions between polymer-

ized antibodies and the SC at the mucosal surface.

The differential pattern of expression was also seen for

heavy chain constant regions of the other immunoglo-

bulin subtypes. Both Cg and Ce had higher expression at

birth and during the end of the switch phase through to the

start of phase 3 and into involution. IgE levels would be

expected to be important in the later stages of lactation, as

this is when the marsupial young would first start eating

herbage and be exposed to parasites. However, differ-

ential regulation was most dramatic in Cg, where

expression increased significantly ( p < 0.01) as the

young left the pouch. Levels of IgG in the brushtail

possum milk remain low until the switch period

(Adamski and Demmer, 2000), whereas in the tammar

wallaby there is also transplacental transfer of IgG and

neonatal serum levels rise quickly in the 20 h post-partum

(Deane et al., 1990). The difference between pouch

young serum IgG levels and the relatively low expression

of IgG (compared to the other immunoglobulins) in the

mammary gland suggests that the tammar may have a

very efficient method of taking up IgG in the neonatal gut.

FcRn, which mediates transepithelial transport of IgG,

has been suggested to play a role in efficiency of IgG

uptake in marsupials (Adamski et al., 2000; Western

et al., 2003). In this study, FcRn expression in the

mammary gland was also affected by differential

regulation. FcRn was increased in virgin animals, around

parturition and in the switch phase. Increases in FcRn

expression in the mammary glands of pigs (Schnulle and

Hurley, 2003), cattle (Mayer et al., 2005) and mice

(Cianga et al., 1999) have been associated with

colostrogenesis. These increases in expression around

parturition and the switch phase reflect not only

expression of IgG in the mammary gland, but also

may represent the role of maternally derived FcRn in

facilitating the uptake of IgG in the gastrointestinal tract

of the neonate, as in mice (Adamski et al., 2000; Western

et al., 2003). Additionally, some motifs associated with

FcRn-binding affinity, transcytosis and serum IgG

persistence in Cg had low conservation in marsupials.

Variation at these motifs has been associated with the

functional differences seen in IgG isotypes (Medesan

et al., 1997), and so this may also be the case in

marsupials.

In this study, the tammar mammary gland expressed

both k and l light chain isotypes. While these subtypes

have been previously identified in both the grey short-

tailed opossum (Lucero et al., 1998; Miller et al., 1999)

and brushtail possum (Belov et al., 2001, 2002a), there

have been no studies that have isolated them in the

tammar wallaby or examined their expression in the

lactating mammary gland of a marsupial. This study

suggests that, unlike the case in camelids (Hamers-

Casterman et al., 1993), tammars can express both types

of light chains in a mature immunoglobulin molecule.

Expression of these light chains in the mammary gland

throughout lactation followed the same differential

regulation pattern seen for the heavy chain constant

regions. The concurrent and similar expression profile

of both subunit components of the immunoglobulin

molecule further strengthens the notion that tammars

and marsupials in general have two periods of increased

immune transfer during their lactation cycle.

A preference for one light chain over the other is often

seen in different mammalian species (Butler, 1997). In

this study, based on the relative frequency of clones in the

mammary gland EST library, there is a preference for l

chains, which is similar to the case in horses, cattle and

sheep (Butler, 1997). This demonstrates a similar

preference to the platypus, where l transcripts made

up over 90% of the mRNA transcripts in a spleen cDNA

library (Johansson et al., 2005). Regardless of the

preference for one light chain over the other, the isolation

of several different Vk and Vl clones in the tammar

mammary gland EST library suggests that a considerable

potential immunoglobulin repertoire is present in the

tammar. In all tammar Vland Vk clones, there was a high

levels of homology in framework regions, especially

FR2, which has been associated with both heavy/light

chain interactions and antigen-binding affinity (Masuda

et al., 2006). However, there was variability in the CDR1

and CDR3 in both Vland Vk, which is a mechanism for

increasing light chain region diversity (Johansson et al.,


2005; Monson et al., 2000; Nowak et al., 2004). Ck and

Cl regions had higher levels of homology than the V

regions, although residues important in heavy/light chain

interactions were not always conserved. This is also the

case in eutherians, where only those residues with the

highest number of contacts between the light chains are

the best conserved (Miller, 1991). This would then

suggest that residues 120, 133, 164 and 179 play an

important role, as these were conserved or had

conservative substitutions in the majority of species

examined. Overall the results of this work suggest that the

tammar wallaby has l and k diversity that is suggestive of

a large potential immunoglobulin light chain repertoire.

Involution in the mammary gland is thought to be

divided into two stages in eutherians—an early and

reversible stage in the first 48 h, followed by an

irreversible stage involving alveolar collapse, the

destruction of the base membranes and the phagocytic

clearance of milk and apoptotic bodies. Many studies

have demonstrated a prominent role for the immune

system in this involution process. Soluble defence

factors and cells that are involved in humoral immunity

are induced in involution to prevent mastitis during this

period of milk stasis. Immunoglobulins are the largest

group of genes up-regulated in involution—in cattle,

levels remain high until late involution; in mice levels

decrease 20 days after forced weaning (Stein et al.,

2004). In this study we observed high expression levels

of many immunoglobulin components and associated

receptors during the early stages of involution in the

tammar mammary gland. Immunoglobulins such as IgG

and IgM are required during involution to facilitate

opsonisation by macrophages and phagocytes, while

IgA may act in the direct sequestration of bacteria

(Clarkson et al., 2004). The results from this study

suggest then that these immunoglobulins may also have

similar roles in the involuting gland of the tammar, but

further work is required to determine if immunoglo-

bulins are important throughout the entire involution

period in the tammar.

5. Conclusion

In conclusion, in this study we have isolated and

characterized the heavy chain constant regions of

immunoglobulin isotypes, their k and l light chains, the

receptor FcRn, J chains and the chemokine CCL28 in

the tammar wallaby. While there were high levels of

conservation of functional areas of these molecules in

the tammar and other marsupials, we did identify some

motifs, which were poorly conserved and may have

potential effects on expression or function of these

immunoglobulins. The tammar wallaby was seen to

have high l and k chain diversity similar to that seen in

eutherians. We have also shown that these immunoglo-

bulins and receptors (including the previously char-

acterized tammar pIgR) display a distinct pattern of

differential regulation in the mammary gland through-

out the lactation cycle. Expression of these immune

components increases during periods of maximum

immune challenge for the young (that is, birth and upon

leaving the pouch). A similar pattern has been

previously described in the brushtail possum for IgA,

IgG (Adamski and Demmer, 1999, 2000), ferritin,

transferrin (Demmer et al., 1999) and FcRn (Adamski

et al., 2000; Western et al., 2003). Together these

findings suggest that differential regulation and

expression of immune components in the mammary

gland throughout lactation is an important strategy for

defence of the vulnerable marsupial young.

Acknowledgements

This work was supported by the CRC for Innovative

Dairy Products, Australia and an Australian Postgrad-

uate Award from the University of Sydney.

Appendix A. Supplementary data

Supplementary data associated with this article can

be found, in the online version, at doi:10.1016/

j.vetimm.2007.07.008.

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