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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 12 6002-6007
Copyright © 2003 by The Endocrine Society

Characterization of a Circulating N-Extended Form of the Thyrotropin-Releasing Hormone-Like Peptide pGlu-Glu-Pro Amide in Human Plasma

A. J. Ruiz-Alcaraz and J. del Rio-Garcia

Department of Pharmacology, Faculty of Medicine, University of Murcia, Espinardo 30100, Murcia, Spain

Address all correspondence and requests for reprints to: J. del Rio-Garcia, Department of Pharmacology, Faculty of Medicine, University of Murcia, Espinardo 30100, Murcia, Spain. E-mail: jdelriog{at}um.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The TRH-like peptides pGlu-Glu-Pro amide, pGlu-Phe-Pro amide, and pGlu-Gln-Pro amide were isolated and identified some years ago, and these peptides have been proven to be present in many tissues and fluids. The presence of TRH-like immunoreactivity distinct from TRH in blood has been observed previously. In the present study, the presence of N-extended forms of TRH-like peptides in plasma has been investigated. Peripheral blood samples of human, rat, and rabbit were obtained and plasma was extracted. The peptides were separated in several steps of chromatography, including gel filtration, cation and anion exchange, and HPLC. The concentrations of the TRH-like peptides in the column fractions were measured by RIA with TRH antibody. The N-extended forms of TRH-like peptides were determined by RIA after trypsin digestion. In human plasma it was observed an N-extended form of TRH-like peptides in substantial concentration. After trypsin and heating, the N-extended forms of TRH-like peptides were rechromatographed on Sephadex G-50. This showed that the TRH-like peptides released have a similar size to TRH. The peptides were then separated by cation exchange chromatography, and the major fraction was unretained, indicating a neutral or acidic nature. Part of the unretained fraction was then chromatographed on anion exchange column in which the major fraction was retained, demonstrating the acidic nature of the peptides. Similar results have been observed in rat and rabbit. The other part of the unretained fraction from cation exchange chromatography of human plasma was purified on HPLC. The results demonstrated that the major component observed by HPLC cochromatographed with synthetic pGlu-Glu-Pro amide. This study represents the first demonstration of a circulating N-extended form of any TRH-like peptide.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PRESENCE OF TRH immunoreactivity has been reported in a series of peripheral tissues, and it was shown that pancreas (1, 2, 3), placenta (2), prostate (4, 5), testis (4), and seminal fluid (6, 7, 8) contained peptides that reacted with TRH antisera. In many cases, however, it appeared that the immunoreactivity did not indicate the presence of the hypothalamic hormone (2, 6). The antibodies used in these studies were specific for the pyroglutamyl residue at the N terminus of TRH and the proline amide residue at the C terminus but were tolerant to the nature of the amino acid in the central position; consequently the observed TRH immunoreactivity could have been due to peptides that were similar in structure to TRH but differed in position 2 of the tripeptide sequence. In this context it was shown by HPLC that rat portal blood contains a number of chromatographically distinct components that react with a TRH antiserum (9), and similarly it has been shown that human seminal fluid contains a TRH homologous peptide that is more hydrophobic than TRH (6).

Three naturally occurring peptides with structures related to TRH were identified some years ago. In these peptides the histidine residue of TRH is replaced by glutamic acid (10), glutamine (11), or phenylalanine (11), and they retain much or all of the immunoreactivity of TRH. Their structures were established by comparison with the corresponding synthetic peptides on HPLC, cochromatography with 3H-labeled synthetic peptides, determination of amino acid composition and sequence, and mass spectrometry (10, 11, 12, 13). These TRH-like peptides were first identified in prostate (10, 14), testis (12), and seminal fluid (11, 15), but they also occur in a range of endocrine tissues and brain (16, 17). Further evidence for the presence of TRH-like peptides in prostate, testis, pancreas, and pituitary has been reported (18, 19, 20, 21, 22, 23, 24, 25), and a TRH immunoreactive peptide in the serum of patients with certain carcinoid tumors has been identified as pGlu-Glu-Pro amide (26).

The TRH-like peptides possess a C-terminal amide group, which is a characteristic feature of many biologically active peptides. This suggests that they are likely to fulfill physiological roles. Because TRH-like peptides occur in substantial quantity in seminal fluid, the possibility was considered that they might play a role in fertility, and recently it has been shown that pGlu-Glu-Pro amide can increase the capacitation of sperm cells (27, 28, 29). In addition, TRH-like peptides produce certain effects in the central nervous system, including increase of locomotor activity and influence on adrenergic transmission (30, 31). The TRH-like peptides pGlu-Phe-Pro amide and pGlu-Glu-Pro amide, administered sc, influence thyroid hormone levels in the circulation (32), and the tissue levels of TRH-like peptides in the thyroid were influenced by thyroid status (33), pointing to a possible role for these peptides in the regulation of thyroid hormones. Furthermore, it was demonstrated the presence of TRH-like peptides in mammary gland and milk being the levels in tumor tissue significantly higher than in normal mammary tissue from the same patients (34).

The presence of TRH-like peptides in a variety of central and peripheral tissues has made necessary the studies on their biosynthetic origin. It was reported that peptides terminating in the sequence Lys-Gln-Glu-Pro amide could be obtained from human semen (35), and it was noted that their sequences corresponded to a region of semenogelin-I, which is a major protein in human seminal fluid. The fact that the sequence Gln-Glu-Pro, possible precursor of the TRH-like peptide pGlu-Glu-Pro amide, was present in the sequence of semenogelin led to the study with molecular biology techniques of the sequence of marmoset semenogelin, a primate whose prostate contained substantial concentrations of the TRH-like peptide. This study showed that the sequence Gln-Glu-Pro from human semenogelin is replaced by Gln-Asp-Gln, which demonstrated that semenogelin is not the precursor of pGlu-Glu-Pro amide (36).

In the present study, we investigated the presence of TRH-like peptides and their N-extended forms in human plasma in comparison with other species such as rat and rabbit.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental tissue

Adult male rats (Sprague Dawley, 200–250 g) or rabbits (New Zealand, 2.5 kg) were given free access to standard chow and water and maintained under controlled conditions of temperature (22 C) and humidity (50%). Rat or rabbit blood was obtained by cardiac puncture under light ether anesthesia (1 and 5 ml/animal, respectively). Human blood was obtained from a vein of the arm of healthy volunteers (5 ml). In each case plasma was obtained after centrifugation (2000 rpm) of the heparin-treated blood and stored at -70 C until used for extraction of peptides.

Extraction of peptides

Plasma (2–3 ml) was homogenized at 4 C in 30 ml of acid acetone (hydrochloric acid: acetone: H2O, 1: 40: 5 vol/vol) to which was added 125I-TRH (approximately 2 x 103 cpm, Perkin-Elmer, Boston, MA) as an internal standard. The suspensions were centrifuged at 2 x 104 rpm, 4 C for 20 min using a Beckman (Palo Alto, CA) J2–21 centrifuge, the supernatants removed by decantation, and the solutions concentrated to dryness in vacuo. The peptides were taken up in 4 ml of 1 M acetic acid and the solutions clarified by microfugation.

RIA of TRH-related peptides

The TRH-like peptides were located in aliquots of column fractions by RIA with TRH antibody. N-extended forms of TRH-like peptides were located in the aliquots by RIA carried out after incubation with trypsin (16 h) and maintained at 150 C for 5 h to complete the cyclization of the N terminal. RIA with TRH antibody was carried out in a total volume of 300 µl by addition of 200 µl of RIA buffer, 50 µl of 125I-TRH, and 50 µl of antibody.

The concentrations of the peptides indicated in the figures have not been corrected for immunoreactivity and therefore represent minimum values. The amounts of TRH immunoreactivity were calculated by comparison with a TRH standard.

The RIA procedure employed has been described in detail (16, 33, 37). The antiserum, which was raised in sheep (38), was used at a final dilution of 1:96,000. The specificity of the TRH antibody used has been described (15, 38). It exhibits a specific requirement for the pyroglutamyl residue at the N terminus of TRH and the proline amide at the C terminus but can accept other amino acids at position 2 of the tripeptide sequence.

Gel exclusion chromatography

Initial purification of plasma peptides was performed by gel exclusion on a column (50 x 1.5 cm) of Sephadex G-50 Superfine in 1 M acetic acid, and fractions (3.1 ml) were collected. The peptides present were detected by RIA with TRH antibody. The elution position of the 125I-TRH marker was determined by counting column fractions using a {gamma}-counter (Cobra model auto-{gamma}, Packard, Meriden, CT).

Cation exchange chromatography

Chromatography was carried out on minicolumns (5 x 0.6 cm) of SP-Sephadex C-25 in the pyridinium form and equilibrated in 50% acetic acid. After addition of the extracts to the columns, the TRH-like peptides were eluted in 50% acetic acid (8 x 0.5 ml), and TRH was then eluted in 0.4 M pyridine in 50% acetic acid (12 x 0.5 ml). 125I-TRH radioactivity was determined on a {gamma}-counter (Cobra model auto-{gamma}, Packard). Aliquots of each fraction was removed and dried in vacuo before determination of TRH-like peptides by RIA.

Anion exchange chromatography

Chromatography was carried out on minicolumn (5 x 0.6 cm) of diethylaminoethyl (DEAE)-Sephadex A-25 prepared by washing with 400 mM sodium phosphate at pH 7.5 and equilibrating in 50 mM sodium phosphate at the same pH. After addition of the sample in 50 mM sodium phosphate to the column, elution was carried out in the same buffer to provide eight 0.5-ml fractions and then in 400 mM sodium phosphate to provide another twelve 0.5-ml fractions. Aliquots of the column fractions were dried in vacuo before analysis by RIA with TRH antiserum.

HPLC

Peptides not retained during cation exchange chromatography were purified by reversed-phase HPLC in 10 mM HCl on a Nucleosil 120–5C18 column (120 x 4 mm, Scharlau Science, Barcelona, Spain). The chromatography was carried out using a 1050 HPLC system (Hewlett Packard, Portland, OR) with a flow rate of 1.5 ml/min and a gradient of 0.5%/min acetonitrile for 6 min followed by 2%/min for 18 min. To ensure the retention of weakly retained peptides, the HPLC column was equilibrated in 10 mM HCl for 20 min before application of the samples, and the solutions were acidified to pH 2 before chromatography. Fractions (0.45 ml) were collected at 0.3-min intervals, and absorbance at 215 nm was recorded continuously. The peptides were located in aliquots of the column fractions by RIA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gel exclusion chromatography of peptides from plasma

The peptides extracted from human plasma (3.0 ml) were purified by gel exclusion chromatography on Sephadex G-50. To detect the presence of N-extended forms of TRH-like peptides, RIA was carried out after incubation with trypsin and heating of the aliquots. A high concentration of TRH-like immunoreactive peptides (1 nmol/ml) released from N-extended forms was observed (Fig. 1Go). These N-extended peptides exhibited much less retention on Sephadex G-50 than 125I-TRH used as internal standard. To confirm the size of the TRH-like peptides obtained after trypsin digestion of the N-extended forms, aliquots of fractions 10–18 of this column were combined, incubated with trypsin, heated, and rechromatographed on a new Sephadex G-50 column. The results showed that the TRH-like immunoreactive peptide obtained coeluted with 125I-TRH (Fig. 2Go).



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  		FIG. 1. Gel exclusion chromatography of TRH-related peptides present in human plasma, demonstrating the presence of N-extended forms of TRH-like peptides. After extraction from the tissue, the peptides were fractionated on a column of Sephadex G50 (fraction size 3.1 ml). The peptides were detected by RIA carried out after trypsin digestion and heating. The arrow indicates the elution position of 125I-TRH.

 


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  		FIG. 2. Gel exclusion chromatography of TRH-like tripeptides released from N-extended forms in human plasma. The tripeptides were liberated from their precursors (fractions 10–18, Fig. 1Go) by the action of trypsin and isolated on a column of Sephadex G50 (fraction size, 2 ml). The peptides were located and their amounts determined by RIA. The arrow indicates the elution position of 125I-TRH.

 
Similar results on N-extended TRH-like peptides were obtained in rabbit and rat plasma in the same assay conditions (Table 1Go).


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  		TABLE 1. N-extended forms of TRH-like immunoreactive peptides in human, rabbit, and rat plasma1

 
Cation exchange chromatography of TRH-immunoreactive peptides release by trypsin from N-extended forms in plasma

Aliquots from the fractions 10–18 of gel filtration of human plasma, which contained N-extended forms of TRH-like peptides, were combined and digested with trypsin, heated, and then chromatographed in a Sephadex SP-C25 minicolumn. The results obtained showed that the major portion of TRH immunoreactivity, released by trypsin from N-extended forms, was not retained on cation exchange chromatography (Fig. 3Go). This implicated that such peptides did not have a positive charge.



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  		FIG. 3. Cation exchange chromatography of TRH-like peptides released from N-extended forms by trypsin, demonstrating their nonretention on the column. The tripeptides were first isolated by gel exclusion chromatography on Sephadex G50 and then fractionated on minicolumns of SP Sephadex C25. The arrow indicates the elution position of 125I-TRH.

 
Very similar results were obtained in rabbit and rat plasma (Table 2Go).


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  		TABLE 2. Cation exchange chromatography of TRH-like peptides released from N-extended forms present in human, rabbit, and rat plasma

 
Anion exchange chromatography of TRH-immunoreactive peptides not retained by cation exchange chromatography

Aliquots from the fractions not retained on cation exchange chromatography (fractions 4–7) of human plasma were combined and chromatographed on DEAE Sephadex A-25. The results showed that the major TRH immunoreactive component was retained (Fig. 4Go), demonstrating the presence of a negative charge in the molecule of the TRH-like peptide. Table 3Go shows the similar results obtained in rabbit and rat plasma.



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  		FIG. 4. Anion exchange chromatography of TRH-like peptides released from N-extended forms by trypsin, demonstrating their retention on the columns. The tripeptides were first isolated by gel exclusion chromatography on Sephadex G50 and then fractionated on minicolumns of SP Sephadex C25 and finally on minicolumns of DEAE Sephadex A25.

 

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  		TABLE 3. Anion exchange chromatography of TRH-like peptides released from N-extended forms present in human, rabbit, and rat plasma1

 
HPLC of TRH-like peptides unretained by cation exchange chromatography of human plasma

The rest of the unretained fraction on cation exchange chromatography of the human TRH-like peptides was combined and chromatographed in HPLC. The results showed a sole component emerging in the same position that synthetic pGlu-Glu-Pro amide chromatographed subsequently (Fig. 5Go).



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  		FIG. 5. HPLC of TRH-like peptides released from N-extended forms by trypsin. After the enzymic digestion, the tripeptides were isolated on a column of Sephadex G50 and minicolumns of SP Sephadex C25. The arrows indicate the elution position of TRH (1 ), pGlu-Glu-Pro amide (2 ), and pGlu-Phe-Pro amide (3 ).

 
DEAE chromatography of the HPLC component from human plasma

To confirm the acidic nature of the TRH-like component observed by HPLC, it was combined and chromatographed on a DEAE minicolumn. Figure 6Go shows that the TRH-like peptide found by HPLC with the same behavior as pGlu-Glu-Pro amide had in fact a negative charge at position 2 of tripeptide.



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  		FIG. 6. Anion exchange chromatography on minicolumns of DEAE Sephadex A25 of the TRH-like peptide released from N-extended forms by trypsin shown in Fig. 5Go, confirming the acidic nature of the peptide.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These results demonstrated the presence of N-extended forms of the TRH-like peptide pGlu-Glu-Pro amide in plasma. This presence has been shown in human and other mammal species such as rat and rabbit. The demonstration of the nature of the TRH immunoreactivity released by trypsin and heating was carried out studying the chromatographic properties of the released peptide on cation and anion exchange chromatography and HPLC. The TRH immunoreactive peptide release by trypsin and heating was chromatographed on SP Sephadex C25 in which it was not retained, demonstrating the absence of any positive charge. This indicated that the peptide released from N-extended forms is not the natural hypothalamic hormone, TRH. The nonretention on SP Sephadex also indicated that the N terminus of the TRH immunoreactive peptide is not an amino group, pointing to the presence of a pyroglutamyl residue at position 1 of the TRH-like peptide after trypsin digestion and heating to complete the cyclization. The TRH immunoreactivity not retained on SP Sephadex was then eluted on reverse-phase HPLC. The results demonstrated that the TRH immunoreactive peptide was eluted in the same position as synthetic pGlu-Glu-Pro amide. After HPLC the peptide was also chromatographed in a DEAE Sephadex A-25 in which it was found to be retained. This result, which confirms the acidic nature of the peptide, and the specificity of the antibody for the amide group at the C terminus of TRH-like peptides and the pyroglutamyl residue at the N terminus, together with the identical chromatographic behavior to synthetic pGlu-Glu-Pro amide, demonstrated that the TRH immunoreactive peptide released from N-extended forms in human plasma is, in fact, the known TRH-like peptide pGlu-Glu-Pro amide.

The presence of TRH-like immunoreactivity, distinct from TRH in blood, was first demonstrated by Sheward et al. (9). These authors showed that the TRH immunoreactivity present in portal blood can be attributed to components distinct from TRH that can be separated by HPLC. Only one of these components chromatographed with the same retention as authentic TRH. Similar results were observed in rat portal (16) and peripheral blood (del Rio-Garcia, J. and D. G. Smyth, unpublished observations). This demonstrated the presence of TRH immunoreactivity distinct from TRH in peripheral blood as well as in portal blood. The presence of the TRH-like peptide pGlu-Glu-Pro amide in human serum has been recently demonstrated (26, 39, 40). The pGlu-Glu-Pro amide has been shown to be present in not only male and female control subjects but also patient with carcinoid tumors in which the serum levels of the peptide were significantly higher. This group also has shown that the levels of pGlu-Glu-Pro amide in postmenopausal women were similar to those observed in premenopausal women, and they provide evidence that this peptide is not secreted by the pituitary gland.

The fact that the N-extended forms of pGlu-Glu-Pro amide found in the amide group points to the possible physiological role in the blood because the amide group is a characteristic feature of many peptide hormones. It has been shown that pGlu-Glu-Pro amide increases capacitation of sperm cells in semen (27, 28, 29). This peptide, administered sc, also increases plasmatic concentrations of thyroid hormones in the rat (33), and the thyroid status of the rat influences the tissular levels of TRH-like peptides in the thyroid (32). On the other hand, it could be possible that the N-extended forms of the tripeptide are the circulating precursors of a shorter active peptide that undergo extracellular processing at the site of action when it is needed. This processing has been described for other peptide hormones such as natriuretic hormone (41).

In conclusion, in human plasma the existence of substantial concentrations of N-extended forms of the TRH-like peptide pGlu-Glu-Pro amide is demonstrated. Similar results have been found also in rabbit and rat plasma.


   Acknowledgments
 
We are very grateful to Professor D. G. Smyth (Department of Pharmacology, University of Murcia, Spain) for his valuable scientific discussion and for providing the TRH-antiserum, generously donated by Dr. H. M. Fraser (Medical Research Council Reproductive Biology Unit, Edinburgh, Scotland, UK).


   Footnotes
 
This work was supported by Grant 00752/CV/99 from the Seneca Foundation (Murcia, Spain).

Abbreviation: DEAE, Diethylaminoethyl.

Received April 18, 2003.

Accepted August 21, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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