Abstract
purpose. RNAs encoding secretoglobin subunits, also known as lipophilins, are found in rabbit lacrimal glands. Some secretoglobins in other species are tissue- and/or sex-specific, whereas other secretoglobins are expressed in a variety of tissues of both sexes. In this study the tissue-specific and sexual dimorphic expression patterns of rabbit lacrimal gland lipophilins AL, AL2, BL, CL, and CL2 were determined.
methods. RNAs from male and female rabbit lacrimal glands were compared in a differential display analysis, and a new rabbit lacrimal gland secretoglobin, lipophilin AL2, was identified. Next, RNAs from male and female rabbit harderian, lacrimal, mandibular, sublingual, and parotid glands and from liver, kidney, pancreas, testis (male), ovary, and mammary gland (female) were isolated, electrophoresed in agarose gels, and transferred to nylon membranes. cDNA probes encoding the lipophilins AL, AL2, BL, and CL/CL2 were hybridized to the RNA in the blots.
results. Rabbit mRNAs encoding the lipophilins AL, AL2, BL, CL, and CL2 were detected only in the lacrimal gland. Lipophilin AL2 mRNA was detected only in male rabbit lacrimal gland.
conclusions. In the rabbit, several lipophilins were expressed only in the lacrimal gland.
The ocular tear film coats the corneal and conjunctival epithelia, providing lubrication and protection for the eye surface. The tear film is an aqueous-mucin gel, covered by a thin lipid layer at the air–fluid interface. Lacrimal glands secrete the bulk of the fluid phase, consisting primarily of water, ions and a variety of dissolved proteins. Goblet cells in the conjunctival epithelium supply many dispersible mucins, proteins with large carbohydrate moieties, whereas corneal and conjunctival epithelial cells express membrane-bound mucins. Meibomian glands embedded in the eyelid margin secrete tear film lipids. The intraocular harderian glands of nonprimate vertebrates also contribute tear proteins and lipids.
Among the proteins present in the aqueous component of tears are members of the secretoglobin family.
1 Secretoglobins are small, multimeric proteins secreted in the mucosa of mammalian epithelial tissues.
2 Uteroglobin represents the founding member of this family,
3 designated SCGB 1A1 (see the phylogenetic tree available through the Human Genome Organisation Gene Nomenclature Committee [HGNC] at http://www.genenames.org/genefamily/scgb_tree.pdf). Homodimers of uteroglobin are present in the uterus, the lung (known as CCSP), and several other tissues.
4 Earlier studies have shown that uteroglobin binds progesterone, inhibits phospholipase A2 and exhibits anti-inflammatory or immunomodulatory properties.
5 6 7
Several investigators have shown that the secretoglobins in saliva and tears are composed of multiple, nonidentical subunits in the form of heterodimers and heterotetramers.
1 8 9 10 In mouse salivary glands, the secretoglobins, known as androgen-binding proteins (ABPs), are composed of three subunits (α, β, and γ), and form α-β and α-γ heterodimers.
11 The corresponding genes are designated
abpa,
abpb, and
abpg. Mouse lacrimal glands express a distinct set of ABP subunits encoded by
abpd,
abpe,
abpz, and
abph.
12 The secretoglobin genes of mouse salivary and lacrimal glands, along with cat salivary gland Fel dI,
13 belong to two branches of the secretoglobin family tree: SCGB 1B and its heterodimer partner designated 1C (see the phylogenetic tree available through HGNC at http://www.genenames.org/genefamily/scgb_tree.pdf). Mouse salivary ABP multimers bind steroid hormones
14 15 and have been proposed to function as pheromones.
16 17
In human lacrimal glands and tears, two different secretoglobin subunits have been identified belonging to the 1D and 2A SCGB branches: lipophilins A and C, also known as SCGB1D1 and SCGB2A1, respectively.
1 In rabbit lacrimal glands, the mRNAs for five lipophilin subunits have been identified (GenBank accession numbers AF308614, AF308617, AF308618, AY063770, and AY303698; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD), belonging to the same 1D and 2A branches of the secretoglobin tree. (Other lipophilin sequences of the 1D and 2A branches have been found in rabbit salivary glands and prostate; GenBank accession numbers AF308615, AF308619 and AF308620.) Rabbit lipophilin proteins are secreted in tears as 17- to 20-kDa covalent-linked heterodimers, which associate as noncovalent heterotetramers of approximately 36.5 kDa.
18 19
Several secretoglobin subunits exhibit sexually dimorphic expression in salivary or ocular glands; examples include cat Fel dI,
20 21 hamster heteroglobin,
10 22 the C3 component of rat prostatein,
8 and mRNAs encoding the mouse ABP-δ and ABP-η.
12 Individual secretoglobin subunits also differ widely in their tissue distribution. Some, such as SCGB1A1 (uteroglobin) and SCGB2A1 (also known as mammaglobin B) in humans, are expressed in a variety of tissues throughout the body; whereas human SCGB1D1 is found primarily in the eye (based on GenBank human EST [expressed sequence tag] database entries, 2007). Likewise in the mouse, secretoglobins ABP-δ and ABP-ζ are expressed in multiple tissues, whereas lacrimal gland ABP-ε and ABP-η, show tissue expression limited to the lacrimal gland.
12
In this study, we characterized the mRNA expression patterns of five rabbit lacrimal gland secretoglobins in RNA blots of male and female rabbit tissues. The lipophilins AL, AL2, and BL (SCGB1D branch) and CL and CL2 (SCGB2A branch) exhibited lacrimal gland–specific expression in the rabbit. In addition, lipophilin AL2 mRNA was expressed only in male rabbit lacrimal glands.
New Zealand (NZ) rabbits (2–3 kg; Bakkom Rabbitry, Viroqua, WI) and Dutch Belted (DB) rabbits (1–2 kg; Birchwood Farms, Red Wing, MN) were treated in accordance with the ARVO Statement for the Care and Use of Animals in Ophthalmic and Vision Research. Male and female rabbits were euthanatized and the following tissues were collected: lacrimal, harderian, mandibular, sublingual, and parotid glands and liver, kidney, pancreas, testis, mammary gland, and ovary. The tissues were frozen in liquid nitrogen and stored at −70°C until needed.
The complete cDNA sequence of lipophilins AL2 and BL were obtained using the rapid amplification of cDNA ends (RACE) technique.
25 cDNA was synthesized with AMV reverse transcriptase (Promega) after priming poly(A)
+ RNA from male and female NZ rabbit lacrimal glands with a sequence-specific oligonucleotide (Sigma Genosys; The Woodlands, TX) for 5′ RACE (lipophilin AL2 and BL), or with oligo-d(T)
17-adapter primer for 3′ RACE (lipophilin BL). Gene-specific PCR primers are listed in
Table 1 ; the adapter primer was 5′-GACTCGAGGATCCAAGC-3′ and the oligo-d(T)
17-adapter primer was 5′-GACTCGAGGATCCAAGC(T)
17-3′.
DNA polymerase (AmpliTaq; Applied Biosystems, Foster City, CA) was used for all amplifications. The PCR reaction mixtures were denatured at 94°C for 2 minutes, then cycled 30 times in a DNA thermal cycler (model 480; Perkin-Elmer, Wellesley, MA) as follows: 94°C for 45 seconds, 52°C for 1 minute, and 72°C for 2 minutes. The last cycle was incubated at 72°C an additional 10 minutes. The PCR products were subcloned into plasmid vector pCR2.1.
Poly(A)+ RNA (2 μg per lane) or total RNA samples (10 μg per lane) were denatured in 50% formamide and 6% formaldehyde in low ionic strength buffer (20 mM phosphate, pH 7.7) at 65°C for 5 minutes. The samples were electrophoresed through a 1.8% agarose gel containing 6% formaldehyde in 20 mM phosphate buffer (pH 7.7). RNA standards (Life Technologies, Rockville, MD) were visualized by staining a portion of the gel with 0.5 μg/mL ethidium bromide. The RNA in the remainder of the gels was capillary transferred to GeneScreen (NEN Life Science Products, Boston, MA) using 10× SSC (1.5 M NaCl, 0.15 M sodium citrate, pH 7.0). Prehybridizations and overnight hybridizations were in 50% formamide at 42°C (39°C for lipophilin BL) according to the manufacturer’s protocol.
The rabbit sequences represented in the lipophilin probes are listed in
Table 2 . Lipophilin clones were digested with appropriate restriction enzymes, the desired fragments were purified in agarose gels, and each fragment was resubcloned before isolation and labeling.
The actin probe was a 1.5-kb mouse α-actin fragment (Stratagene, La Jolla, CA). The G3PDH probe was 1-kb fragment of human glycerol-3-phosphate dehydrogenase (BD-Clontech, Palo Alto, CA). DNA fragments were random prime-labeled with [α-32P]dCTP (NEN Life Science Products) using the a gene labeling system (Prime-a-Gene; Promega). The labeled probes were denatured in a boiling water bath for 5 minutes and added to fresh hybridization solution at 1 to 6 × 106 cpm/mL for overnight incubations.
The blots were washed several times in 2× SSC, 1% SDS at 60°C (55°C for mouse actin; 52°C for lipophilin BL) and finally in 0.2× SSC at room temperature. The blots were exposed to XAR film (Eastman Kodak Co., Rochester, NY) for 2 to 4 hours (lipophilins AL and CL2) or to XAR film with an intensifying screen at −70°C for approximately 1 week (lipophilins AL2 and BL). After each hybridization, the blots were stripped in boiling 0.1× SSC with 1% SDS.
We compared mRNA sequences of male and female rabbit lacrimal glands using DDRT-PCR. From male lacrimal glands, we isolated a sequence fragment encoding a previously unidentified rabbit lipophilin mRNA, predicted to encode a SCGB1D protein subunit. We obtained the full-length sequence of the new lipophilin, which we named lipophilin AL2 (GenBank accession no. AY063770). Two other members of the SCGB1D branch had already been identified from rabbit lacrimal glands: lipophilins AL and BL. We also obtained a full-length sequence for lipophilin BL, which included the 5′ untranslated region (GenBank accession no. AY303698).
We compared the cDNA sequence of lipophilin AL2 with other rabbit lipophilin sequences of the SCGB1D branch. The 3′ untranslated region of lipophilin AL2 contains a 40-base sequence element, bases 410 to 450, conserved among SCGB1D mRNAs of human, rabbit and rat
(Fig. 1) .
The deduced amino acid sequence of lipophilin AL2, bases 73 to 363, encodes a 96 residue preprotein. SignalP predicts cleavage of a signal peptide after serine 21, yielding a 75-amino-acid mature protein subunit with a calculated pI of 4.8.
We aligned the deduced amino acid sequence of lipophilin AL2 with those of other rabbit and human members of the SCGB1D branch
(Fig. 2) . For reference, we included an alignment of rabbit and human members of the SCGB2A branch. Rabbit lacrimal gland lipophilins CL and CL2 were available in sequence databases. Note that each predicted mature subunit contains three conserved cysteine residues, available to participate in disulfide bond formation in secretoglobin heterodimers or heterotetramers.
To address sex and tissue specificity of the rabbit lacrimal gland lipophilins, we hybridized labeled cDNA fragments of lipophilins AL, BL, and CL2 to RNA blots of NZ rabbit male and female tissues
(Fig. 4) . For this experiment, we specifically collected several salivary glands from one male and one female NZ rabbit, since other lipophilins (AS and CS) have been identified in rabbit salivary glands (see
Fig. 2legend). Lipophilins AL, BL, and CL2 hybridized to RNA from male and female lacrimal glands as found previously for both DB and NZ rabbits
(Figs. 3 4) . None of the lacrimal gland lipophilin sequences hybridized with RNA from any of the other rabbit tissues tested
(Fig. 4) . Unexpectedly, lipophilin AL2 did not hybridize (data not shown) to the lacrimal gland RNA of the adult male NZ rabbit, from which we isolated the tissues for the RNA blot depicted in
Figure 4 . Actin and G3PDH control hybridizations demonstrated that each lane of the RNA blots contained intact RNA (data not shown).
The lipophilin CL2 cDNA fragment likely cross-hybridized with lipophilin CL. To address whether lipophilins CL and CL2 were each expressed in male and female lacrimal glands, we amplified male and female rabbit lacrimal gland cDNA with sequence-specific primers. Lipophilin CL primers yielded a 197-base product and lipophilin CL2 primers a 204-base product, each from cDNAs generated from male and female NZ rabbit lacrimal glands (data not shown). Mixed primer pairs did not yield amplified products.
We also tested the tissue specificity of rabbit lacrimal gland lipophilins in an RNA blot of DB harderian, lacrimal, and salivary glands; liver; and pancreas (data not shown). The lacrimal gland lipophilins AL, AL2, BL, CL, and CL2 were expressed only in rabbit lacrimal gland tissues in both DB and NZ rabbits.
Rabbit lacrimal glands expressed mRNAs for five secretoglobins belonging to the 1D and 2A branches: lipophilins AL, AL2, and BL (1D) and lipophilins CL and CL2 (2A). Individual rabbit secretoglobins exhibited sequence conservation across species in the protein coding regions. In addition, a 40-base motif in the 3′ untranslated region of SCGB1Ds was 84% identical among rabbit, human, and rat sequences. Remarkable conservation of the untranslated regions of secretoglobins has been noted previously for members of the SCGB1D branch in rat
31 and hamster
10 and for mouse secretobglobins known as ABPs.
12 32
In RNA blots of several different rabbit tissues, we found expression of each of the rabbit lipophilins AL, AL2, BL, CL, and CL2 only in lacrimal glands. We did not find evidence for the expression of these lipophilins in rabbit harderian, mandibular, sublingual, parotid, or mammary glands or in heart, liver, kidney, pancreas, ovary, or testis.
We have observed a similarly restricted secretoglobin expression pattern in the mouse. In previous work, we found lacrimal gland–specific expression of mRNAs encoding several mouse secretoglobins, known as ABP-δ, -ε, -ζ, and -η.
12 In RNA blots of mouse eye and salivary glands, we documented the expression of the corresponding genes
abpd,
abpe,
abpz, and
apbh only in lacrimal glands. Although RNA blotting data suggested restricted expression of the mouse lacrimal gland secretoglobins, EST data implied broader tissue distribution of two of these lacrimal gland secretoglobins: the genes
abpd and
abpz.
Expression patterns for individual secretoglobin subunits differ widely. In contrast to the apparent restricted expression pattern we observed for rabbit lacrimal gland lipophilins and some mouse ABPs, there is evidence that several secretoglobins in various species are expressed in both ocular glands and other tissues. In the rat, lacrimal gland and prostate express the mRNA and protein product for the C3 component of prostatein.
8 33 Hamster heteroglobin oligonucleotide probes hybridize to RNA from lacrimal, parotid, and submaxillary glands.
10 In the cat, monoclonal antibodies to Fel dI indicate that this allergen is secreted in cat tears as well as saliva.
34 In the NOD mouse, PCR amplification studies have shown that both lacrimal and salivary glands express LGP10 (77% identical with ABP-η), identified as a Sjögren’s syndrome autoantigen.
35 (Note that mouse salivary glands also express a distinct set of secretoglobin subunit sequences, which include
abpa,
abpb, and
abpg.
11 )
In human lacrimal glands, Zhao et al.
36 identified two secretoglobin subunits, SCGB1D1 and SCGB2A1, and reported broad tissue expression for the genes encoding both subunits. Human ESTs for SCGB2A1 were found in the eye and in a variety of other tissues, including brain, pancreas, thyroid, kidney, muscle, mammary gland, ovary, uterus, prostate, and testis. In contrast, human ESTs for SCGB1D1 have been found only in the eye, probably corresponding to lacrimal gland mRNA expression (based on GenBank human EST database entries, 2007). Different detection methods may be responsible for some of the contradictory findings.
There is precedent in the literature for either broad or highly restricted tissue expression patterns of individual secretoglobin subunits. Although our rabbit RNA blots may have missed some low-level expression, we found limited expression, confined to the lacrimal glands, of each of five rabbit secretoglobins: lipophilins AL, AL2, BL, CL, and CL2.
What functions might these diverse sets of small proteins fulfill? Molecular recognition could provide a plausible explanation for the variability observed in individual secretoglobin subunit sequences and protein multimer composition. Many secretoglobins are expressed in the mucosa at the interface of the organism with its environment. For example, lacrimal gland secretoglobins in tears cover the exposed ocular surface; salivary gland secretoglobins reside in the saliva that coats the oral cavity and constitutes the first contact with ingested substances; lung epithelial cells secrete secretoglobins that inhabit the bronchial mucosa, the interface between inhaled substances and the respiratory system.
Several possible roles for secretoglobin proteins are consistent with their individual compositional variability and tissue distribution. Putative roles include communication with the external world, either in a form of defense as a constituent of immune recognition systems,
41 42 and/or in signaling with other organisms.
43 In support of these conjectures, uteroglobin/CCSP exhibited anti-inflammatory properties,
4 and rodent salivary secretoglobins were suggested to function as pheromones or in odorant recognition.
16 17 The propensity of secretoglobins to bind small hydrophobic molecules, such as steroid hormones, may constitute an integral aspect of their function.
5 44 45
There are tissue-specific and sex-specific gene expression differences among members of the secretoglobin family, within species as well as across species. In this study, we characterized five rabbit lacrimal gland–specific lipophilins. These secretoglobins exhibited sequence homology and subunit structure similarities to the secretoglobins found in human lacrimal glands.
Supported by a grant from HealthPartners Research Foundation, Minneapolis, MN.
Submitted for publication November 21, 2007; revised February 15, 2008; accepted May 9, 2008.
Disclosure:
S.G. Remington, None;
J.M. Crow, None;
J.D. Nelson, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “
advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Clone/Accession No. | Reverse Transcription Primer* | Bases | PCR Primer* | Bases |
5′ RACE | | | | |
Lipophilin AL2/AY063770 | 5′-AAGCAAGATGTTGAAACC-3′ | 417–434 | 5′-AGATAGTAGAAAGCAAGGC-3′ | 363–381 |
Lipophilin BL/AY303698 | 5′-GAAAGCAATTATTTATTAAATC-3′ | 507–528 | 5′-CATCTCAAATCATGTGTTGTG-3′ | 486–506 |
3′ RACE | | | | |
Lipophilin BL/AY303698 | | | 5′-TCACTTTCCTTGCATTCCTC-3′ | 440–459 |
Lipophilin Probe | Bases | GenBank Accession No. |
AL (SCGB1D) | 115–407 | AF308614 |
AL2 (SCGB1D) | 315–454 | AY063770 |
BL (SCGB1D) | 459–532 | AY303698 |
CL2 (SCGB2A) | 296–503 | AF308618 |
The authors thank Melanie Lind-Ayres and Cari Steelman for the early characterization of rabbit secretoglobin sequences.
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