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Rapidly evolving marmoset MSMB genes are differently expressed in the male genital tract
© Lundwall et al; licensee BioMed Central Ltd. 2009
Received: 4 July 2009
Accepted: 9 September 2009
Published: 9 September 2009
Beta-microseminoprotein, an abundant component in prostatic fluid, is encoded by the potential tumor suppressor gene MSMB. Some New World monkeys carry several copies of this gene, in contrast to most mammals, including humans, which have one only. Here we have investigated the background for the species difference by analyzing the chromosomal organization and expression of MSMB in the common marmoset (Callithrix jacchus).
Genes were identified in the Callithrix jacchus genome database using bioinformatics and transcripts were analyzed by RT-PCR and quantified by real time PCR in the presence of SYBR green.
The common marmoset has five MSMB: one processed pseudogene and four functional genes. The latter encompass homologous genomic regions of 32-35 kb, containing the genes of 12-14 kb and conserved upstream and downstream regions of 14-19 kb and 3-4 kb. One gene, MSMB1, occupies the same position on the chromosome as the single human gene. On the same chromosome, but several Mb away, is another MSMB locus situated with MSMB2, MSMB3 and MSMB4 arranged in tandem. Measurements of transcripts demonstrated that all functional genes are expressed in the male genital tract, generating very high transcript levels in the prostate. The transcript levels in seminal vesicles and testis are two and four orders of magnitude lower. A single gene, MSMB3, accounts for more than 90% of MSMB transcripts in both the prostate and the seminal vesicles, whereas in the testis around half of the transcripts originate from MSMB2. These genes display rapid evolution with a skewed distribution of mutated nucleotides; in MSMB2 they affect nucleotides encoding the N-terminal Greek key domain, whereas in MSMB3 it is the C-terminal MSMB-unique domain that is affected.
Callitrichide monkeys have four functional MSMB that are all expressed in the male genital tract, but the product from one gene, MSMB3, will predominate in seminal plasma. This gene and MSMB2, the predominating testicular gene, have accumulated mutations that affect different parts of the translation products, suggesting an ongoing molecular specialization that presumably yields functional differences in accessory sex glands and testis.
Human beta-microseminoprotein (MSMB) is synthesized from a gene located on chromosome 10, which has recently attracted much attention since genome wide association studies identified it to be connected with prostate cancer susceptibility [1, 2]. It is an 11-kDa non-glycosylated protein that is expressed in many tissues, but the concentration is particularly high in prostate secretion . At ejaculation MSMB is transferred with other prostate-secreted components to the seminal plasma, where it has a concentration in the range of 0.5-0.9 mg/ml in young healthy males . The protein is synthesized as a precursor of 114 amino acid residues and contains a signal peptide that is removed during secretion to yield the mature protein of 94 residues, something that is also reflected in its alternative name: prostate-secreted protein of 94 amino acids (PSP94) . Recent NMR studies show that MSMB has a unique structure, with an extended configuration, consisting of a four-stranded Greek key-motif and an exclusive domain of two two-stranded beta-sheets . The only other protein that is assumed to have a similar structure is the newly identified PC3-secreted microprotein (PSMP): a protein that is highly expressed in the prostate cancer cell-line PC3 . The function of MSMB is not yet known, but it forms very strong bi-molecular complexes with cysteine-rich secretory protein-3 (CRISP3) in seminal plasma and PSP94-binding protein (PSP-BP) in blood serum [8, 9].
Phylogenetic studies show that MSMB is present in all this far analyzed vertebrate species and also in the chordate amphioxus [10, 11]. The protein displays a very rapid evolution, as revealed by the low conservation of the primary structure between species: e.g., only 45% of the residues are identical in human and rat MSMB . However, all vertebrate MSMB molecules seem to carry 10 conserved Cys that stabilize the 3D structure by forming 5 disulphide bonds . In the chordate amphioxus, one of these disulfides is missing .
We have previously shown that some New World monkeys, e.g. the closely related cotton top tamarin (Saguinus oedipus) and common marmoset (Callithrix jacchus) of the primitive Callithricidea family, carry several MSMB in their genomes, something that is in contrast to most other vertebrate species, which carry a single MSMB . More recently, we cloned and sequenced 5 MSMB from a cotton-top tamarin genomic library . We concluded that 2 of them were pseudogenes, as one of them, MSMB4, had a deletion that shifted the reading frame and lead to premature termination, and the other, MSMB5, had the features of a processed pseudogene. The remaining three genes, MSMB1, MSMB2, and MSMB3 appeared to be functional from a structural point of view. It was not possible to investigate MSMB transcripts in tamarin tissues due to lack of material, but promoter analysis using luciferase reporter in monkey kidney COS cells showed that only MSMB2 displayed an activity that was comparable with that of human MSMB.
In this study we have extended our investigation of MSMB in the common marmoset in order to physically map the genes at the postulated MSMB locus and to analyze the relative expression of the genes in the male genital tract.
Presently, the HUGO gene nomenclature committee does not provide official gene symbols to genes that are specific to non-human primates. In our earlier publications on cotton-top tamarin MSMB, we used gene names that were based on clone names. As these names clearly do not agree with Hugo's gene naming rules, we have decided to adopt a new nomenclature that is based on the genes' location on the chromosome. The new gene symbols are given with the old symbols written within parenthesis as follows: MSMB1 (mspA), MSMB2 (mspE), MSMB3 (mspJ), MSMB4 (mspB) and MSMB5 (mspH).
The June 2007 Callithrix jacchus draft assembly, produced at the Washington University School of Medicine, St Louis, was probed with the sequence of the human MSMB transcript using the program BLAT, available through the University of California, Santa Cruz, Genome Bioinformatics Site . DNA sequences of the housekeeping genes GAPDH and CSTB were identified by the same method using the human orthologs. The DNA contigs that were identified to contain MSMB or housekeeping gene sequences were then analyzed further using EMBOSS Tools  and the program package Vector NTI, which is freely available through Invitrogen's webpage .
RNA isolation and cDNA synthesis
Prostate, seminal vesicles and testis from a common marmoset, kept in captivity at the German Primate Center in Göttingen, Germany, were recovered and frozen in liquid nitrogen immediately post-mortem and then stored at -80°C until further processing took place. Samples consisting of, 0.09 g prostate or 0.05 g seminal vesicle tissue were homogenized in 1.5 ml Trizol reagent (Invitrogen, Stockholm, Sweden) using a polytron homogenizer (Kinematica Inc, Lucerne, Switzerland). In the same way 0.36 g of testis tissue was homogenized in 6 ml Trizol reagent. RNA extracts were prepared according to the protocol provided with the Trizol reagent. Before cDNA synthesis, samples of 3.3 μg of total RNA were incubated for 30 min at 37°C with 1 unit of RNase-free DNase (Fermentas Sweden, Helsingborg, Sweden) in 10 μl of 10 mM Tris-HCl, pH 7.5, 2.5 mM MgCl2 and 0.1 mM CaCl2, to which 0.5 μl (20 units) of Ribolock RNase inhibitor (Fermentas) was added. To terminate the digestion, 1 μl of 25 mM EDTA was added to the samples, which subsequently were incubated for 10 min at 65°C. Each sample was then supplemented with 1 μl containing 100 pmol of oligo(dT)18, heated for 5 min at 65°C, cooled on ice and subjected to a collect spin. To the samples were then added 4 μl of 5 × reaction buffer (250 mM Tris-HCl, pH 8.3, 250 mM KCl, 20 mM MgCl2 and 50 mM DTT), 0.5 μl (20 units) of Ribolock, 2 μl of 10 mM dNTP and 1 μl (200 units) of RevetAid M-MuLV reverse transcriptase (Fermentas). Control samples were prepared in parallel by omitting the reverse transcriptase. The first strand cDNA was synthesized by incubating the samples for 1 h at 42°C. Finally, the samples were diluted with 180 μl of ultra pure water and then stored at -20°C before further analyses.
Nucleotide sequences of PCR primers.
Primer sequence (5' to 3' orientation)
The specificity of primers was confirmed by PCR on marmoset genomic DNA followed by DNA sequencing. The PCR was run as above, but the RNA was replaced with 10 ng of genomic DNA. Material from 5 PCR reactions was pooled and purified using Jetquick (Genomed, SAVEEN Werner AB, Malmö, Sweden). The DNA concentrations were estimated following electrophoresis by comparing the staining intensity of purified PCR fragments with that of the DNA-bands in the MassRuler DNA ladder. Sequencing reactions were done with 40 ng of DNA template and 4 pmol of diluted PCR primer in a total volume of 20 μl, using the Big Dye Terminator Ready Reaction Premix diluted 1:4 and following protocols provided with supplier of the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Stockholm, Sweden). The DNA sequencing was done on an ABI 3130 DNA Analyzer (Applied Biosystems) as a service by the Clinical Chemistry Department at University Hospital MAS, Malmö, Sweden.
The cDNAs that were synthesized for semi quantitative RT-PCR were also analyzed by real-time PCR in the presence of SYBR Green. In MicroAmp Optical 384-Well Reaction Plates (Applied Biosystems), 10 μl reactions were set up by addition of 2 μl of primer mix (containing reverse and forward primers at 5 μM), 3 μl of diluted cDNA template, and 5 μl of Fast SYBR Green Master Mix (Applied Biosystems). The plate was then sealed with MicroAmp Optical Adhesive Film and real-time PCR was performed using the 7900HT Fast Real-Time PCR System (Applied Biosystems). Each primer pair was run on quadruple samples at different concentrations by serially diluting the templates between 5 and 25 times to yield at least 8 recordings for each gene in each tissue. The real-time PCR was run with the Fast SYBR Green protocol using the following cycling conditions: an initial activation step at 95°C for 20 s, followed by 40 cycles of denaturation at 95°C for 1 s and annealing and extension at 60°C for 20 s. The generated data was analyzed with the Sequence Detection System 2.3 software that is provided with the instrument. Cycle threshold (CT) values were calculated automatically and then slightly adjusted manually to accommodate all samples in their exponential phase.
Primer pair performance.
Relative efficacy (%)
The relative concentration of different MSMB transcript in a tissue was calculated from the difference in CT, i.e. ΔCT, between the endogenous reference, which was the mean CT value of the primer pair generating the lowest CT at a given template dilution, and all measured values at this dilution. The relative transcript levels were obtained by exponentially transforming ΔCT values to 2-ΔCT and the mean values were calculated with one standard deviation . For comparison of MSMB expression in different tissues, ΔCT was calculated as the difference in CT between the mean values of MSMBs and the housekeeping genes GAPDH and CTSB. The sum of 2-ΔCT for all four MSMBs in each tissue was calculated and then used to compute the ratio of MSMB transcripts in testis, seminal vesicles and prostate.
Identification of 5 marmoset MSMB genes
Sequence similarity between common marmoset and cotton-top tamarin MSMB genes.
Molecular properties of the marmoset MSMB genes and proteins
Sizes of marmoset MSMB genes.
Conservation of common marmoset MSMB genes.
Conservation of coding nt/whole gene (%)
The predicted translation products from the different MSMB genes are almost identical in molecular mass and differ by only 0.1 kDa, despite that MSMB1 has only 93 amino acid residues: one shorter than the other proteins. Their calculated isoelectric points vary from acidic for MSMB1, pI 4.9, to slightly alkaline for MSMB2, pI 8.1, with MSMB3, pI 7.2, and MSMB4, pI 6.5, located in between.
Expression in the male genital tract
RNA samples from marmoset prostate, seminal vesicle and testis were analyzed with real time RT-PCR in order to gather detailed information on levels of MSMB transcripts in the male genital tract. The PCR reactions were made with different template dilutions, equivalent to 2 to 10 ng of total RNA. This generated CT-values in the range of 13-31, where the lowest values were obtained with 10 ng of prostate RNA and the highest with 2 ng of testis RNA. The negative controls, i.e. samples without reverse transcriptase, were run with undiluted material equivalent to 50 ng of RNA. Around half of the controls yielded CT-values ranging from 33 to 36, but for the remainder there was no detectable signal. The difference in CT value between 5 times diluted samples, equivalent to 10 ng of RNA, and the matching controls was in the range of 5.8-22.6, which suggests that there is no influence of unspecific signals during the measurement of samples.
Relative expression of marmoset MSMB genes.
exon 2 and 3 primers
exon 3 and 4 primers
3.8 × 10-3
1.1 × 10-3
7.6 × 10-3
1.1 × 10-3
6.9 × 10-3
0.5 × 10-3
8.5 × 10-3
1.9 × 10-3
1.8 × 10-2
0.6 × 10-2
1.6 × 10-2
0.4 × 10-2
4.4 × 10-2
0.8 × 10-2
7.2 × 10-2
0.8 × 10-2
1.1 × 10-2
0.6 × 10-2
1.6 × 10-2
0.2 × 10-2
1.4 × 10-2
0.6 × 10-2
3.6 × 10-2
0.4 × 10-2
We have previously shown by Southern blotting that the common marmoset and the cotton-top tamarin have the same, or almost the same, number of MSMBs . This is now confirmed by the demonstration of 5 MSMBs in the common marmoset that are orthologous with the 5 MSMBs in the cotton-top tamarin . In addition, we identified a unique duplication in the marmoset MSMB1, which has created a new potential transcription initiation site around 2.7 kb upstream of the "normal" start site. Whether this new site is used for initiation of MSMB1 transcription or should be considered as a truncated pseudogene remains to be seen. In earlier studies on the cotton top tamarin it was not possible to determine whether the MSMBs, excluding the processed pseudogene MSMB5, are situated at a single genetic locus, but from the location of homologous regions in two of the genes it was speculated that there probably is an MSMB located around 20 kb downstream of MSMB3 . In this study on the common marmoset it was found that there is indeed a gene located 20 kb downstream of MSMB3, but also another gene located 32 kb upstream. These three genes MSMB2, MSMB3 and MSMB4 constitute a unique MSMB locus that, according to the homology with human chromosome 10, is separated by several Mb from the MSMB locus containing MSMB1, which is conserved in the human and the mouse genomes. Presumably, the functional callitrichine MSMB have evolved by three rounds of duplication. The first presumably involved a duplication that yielded MSMB1 and a precursor to the genes at the unique second MSMB locus. We have previously shown that MSMB3 and MSMB4 are closely related . It is therefore likely that a second duplication yielded MSMB2 and a precursor to these two genes. Finally, a third duplication yielded MSMB3 and MSMB4.
Translated exon sequences of the marmoset MSMBs are more dissimilar than their flanking introns, something that was previously observed also in the cotton-top tamarin . Furthermore, most mutations of translated nucleotides also lead to amino acid replacements. This suggests that MSMBs in the callitrichine monkeys are subjected to an accelerated evolution. Analysis of mutated nucleotides in genes at the unique MSMB locus show that MSMB2 has accumulated mutations in exon 3 and the terminal half of exon 2, whereas MSMB3 is mostly affected in exon 4. This pattern of mutation overlaps with the domain structure of MSMB, such that in MSMB2 it is the first, Greek key, domain that is affected, whereas in MSMB3 it is the MSMB-unique second domain that is affected. The most reasonable interpretation of this phenomenon is that an evolutionary pressure has lead to specialization of the two genes. In contrast, the very few mutations detected in MSMB4 suggest that this gene is not under similar high evolutionary pressure. In fact, in an earlier study we demonstrated that the cotton-top tamarin MSMB4 is a pseudogene, something that could indicate that this gene is subjected to an ongoing purifying selection .
The studies on the expression show that all four functional marmoset MSMB are transcribed in several different cell types in the male genital tract. However, the MSMB concentration in the prostate, seminal vesicles and testis is very different, as can be seen when transcript levels are normalized with housekeeping genes. The level in the seminal vesicles is around 1% of that in the prostate and the level in the testis is even lower, by another two orders of magnitude. From this it can be concluded that almost all MSMB in seminal plasma originates from the prostate, with only minor contribution from seminal vesicles and testis. In the prostate, MSMB3 is clearly dominating, with the remaining three molecular species each contributing a few percent to the total MSMB transcript pool. A similar situation is also found in the seminal vesicles, which are dominated by MSMB3 and with only minor contribution from the other MSMB genes. In a previous investigation, we analyzed common marmoset seminal plasma with isoelectric focusing and demonstrated a predominating molecular species of MSMB with a pI value that was estimated to 7.3 . This value is very close to the theoretically calculation of 7.2 for MSMB3. Thus, the high transcript level in the accessory sex glands is also reflected in a high protein concentration in seminal plasma. The isoelectric focusing also demonstrated two minor molecular species of MSMB, with pI of 6.6 and 4.9. These figures agree with the calculated pI values of 6.5 for MSMB4 and 4.9 for MSMB1, suggesting that the transcripts of these genes are also translated. In contrast, the isoelectric focusing did not display any MSMB molecule that would agree with a pI of 8.1; the calculated value of MSMB2. Whether this is due to poor translation of the MSMB2 transcript, instability of the translation product or other reasons, e.g. posttranslational modification, remains to be determined.
The same predominance of MSMB3, as seen in the accessory sex glands, was not observed in the testis. Instead it was MSMB2 that generated around half of the transcripts, which is interesting and could suggest that MSMB3 only display high transcript levels in the accessory sex glands, whereas in other organs MSMB2 or one of the other gene products are dominating. This is in line with the previous experiments using monkey kidney COS cells, in which only cotton-top tamarin MSMB2 displayed activity comparable with human MSMB in luciferase reporter assays . Perhaps the cell and tissue specific difference in relative expression between MSMB3 and MSMB2 is mirroring the above mentioned putative specialization with accelerated evolution of either the Greek key domain or the MSMB-specific domain. This very interesting aspect could presumably be analyzed in more detail in the future, when once the function of MSMB is known.
The common marmoset has orthologes of all MSMB, previously identified in the cotton-top tamarin, suggesting that multiple MSMB is a property of all Callitrichine monkeys. Transcripts of MSMB1, MSMB2, MSMB3 and MSMB4 are present in both testis and accessory sex glands, but the level in the prostate is around 100 times higher than in the seminal vesicles and 10,000 times higher than in the testis. One gene, MSMB3, accounts for more than 90% of the transcripts in the prostate and the seminal vesicles and yields almost all beta-microseminoprotein in seminal plasma. Marmoset MSMB displays rapid evolution, as revealed by the lower conservation of translated nucleotides compared to introns and flanking DNA. MSMB3 and the predominant gene in the testis, MSMB2, have accumulated mutations that affect different domains of beta-microseminoprotein, suggesting a specialization of these genes which might indicate different function of MSMB in accessory sex glands as compared to testis.
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