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Applied Biochemistry and Microbiology

, Volume 55, Issue 1, pp 13–20 | Cite as

Spontaneous Proteolytic Processing of Human Recombinant Anti-Mullerian Hormone: Structural and Functional Differences of the Molecular Forms

  • A. Ya. RakEmail author
  • A. V. Trofimov
  • E. A. Protasov
  • S. V. Rodin
  • A. V. Zhahov
  • Ya. A. Zabrodskaya
  • A. M. Ischenko
Article
  • 34 Downloads

Abstract

The technology for the production of highly purified human recombinant anti-mullerian hormone (AMH)—a potential antitumor agent for the treatment of certain types of malignant neoplasms—is described. It was found that spontaneous proteolytic processing of the hormone is possible during the storage of AMH preparations under physiological conditions. This leads to the formation of C-terminal homodimer of AMH (activated form) and, later, to an inactive state during the further proteolysis. Sites at which spontaneous processing of the hormone molecule occurred during prolonged storage with the formation of active and inactive fragments were identified. The structural and functional differences in the molecular forms of the C-terminal fragment contained in the preparations are analyzed.

Keywords:

anti-mullerian hormone AMH chromatography MALDI mass-spectrometry monoclonal antibodies recombinant protein proteolysis 

INTRODUCTION

Anti-Muller hormone (AMH) is a substance that inhibits mullerian ducts (a Mullerian inhibitory substance, MIS). It is one of the least-studied glycoproteins of the TGF-β superfamily. It is both an embryonic and a postnatal regulator of the development and functioning of the mammalian reproductive system [1]. Recombinant AMH (rAMH) also has antitumor activity against the cells of a number of human tumors [2, 3]. The AMH molecule with a mass of about 140 kDa is a homodimer, both monomers of which contain in their sequence a proteolytic cleavage site located between the Arg427 and Ser428 amino acid residues. Specific proteolysis of the full-length hormone molecule at this site leads to its splitting into N- and C-terminal homodimers with molecular masses of about 115 and 25 kDa, respectively [1].

Under physiological conditions, N- and C-terminal dimers of AMH form a noncovalently associated complex after proteolysis. It is known that, unlike the N-terminal, the C-terminal homodimer of AMH is biologically active [4]. It is suggested that in vivo proteolysis of AMH occurs in the process of hormone biosynthesis or under the action of furin or plasmin when it enters the tissues and interacts with specific AMH receptors of type I and type II (MISRI and MISRII, respectively) [5, 6, 7].

The purpose of the work is to obtain and characterize highly purified preparations of rAMH and its active form, the C-terminal fragment.

MATERIALS AND METHODS

Culture fluid of CHO (American Type Tissue Collection, United States) cells transfected with the human AMH gene (CHO-MIS #26 producer strain) was used for the preparation of pAMH [8]. The cells were cultured in a Wave Bioreactor 20/50 System reactor (GE Healthcare, United States) in a serum-free CDM4CHO medium (HyClone, United States). The culture fluid was separated by centrifugation, clarified, and used to isolate a full-sized rAMH, from which derivatives of the hormone and its C-terminal fragment were obtained.

Various types of murine monoclonal antibodies (State Research Institute of Highly Pure Biopreparations, FMBA of Russia) and variants of test systems developed on their basis were used to study the properties of rAMH and its derivatives. ACMIS-3 antibodies and ACMIS-4 (ACMIS-4-Px) antibodies conjugated with horseradish peroxidase recognized different epitopes of the c-AMH C-terminal fragment, but they did not interact with the full-length hormone. In this regard, an enzyme immunoassay test system created on the basis of the antibodies (ACMIS-3)–rAMH– (ACMIS-4-Px) (I) was used for the quantitative determination of molecules containing a rAMH C-terminal fragment in a variant of a heterogeneous sandwich enzyme-linked immunosorbent assay (ELISA).

M2 antibodies, which recognize only full-size rAGH and its derivatives containing the N-terminal fragment, were used to isolate and purify full-length pAMH in series with ACMIS-1 antibodies. They were also used to detect the N-terminal fragment of rAMH in direct solid-phase ELISA.

The biological activity of rAGH preparations was assessed by the ability to bind to the extracellular part of the specific AMH receptor MISRII [8]. For this purpose, a special enzyme immunoassay system was developed, the key component of which was the previously obtained recombinant chimeric protein MI-SRII + Fc; it consists of the extracellular part of MISRII and the Fc fragment of human immunoglobulin IgG1. I4 antibodies specific for the human IgG1 immunoglobulin Fc fragment and ACMIS-4-Px peroxidase conjugate were also used in the test system. At the first stage of analysis with this test system, I4 antibodies were sorbed (1.5 μg/mL in 20 mM borate buffer, pH 8.0) and added to the wells. They served as fixing antibodies for the MISRII + Fc chimeric construct, ensuring the correct orientation of its receptor-specific part in the direction of the ligand due to its specificity for the IgG1 Fc fragment. At the next stage, a solution of the chimeric MISRII-containing construct (100 ng/mL) was added to the wells as a specific acceptor for the tested rAMH. After the test sample was applied, pAMH connected with receptor was detected with the ACMIS-4-Ph antibody conjugate (Sigma, United States). The enzyme immunoassay was recorded by the standard method at 450 nm with tetramethylbenzidine and BioRad Reader Model 680 microboards (BioRad, United States). Schematically, the test system to determine the biological activity of rAMH can be described as I4–(MISRII + Fc)–rAMH–(ACMIS-4-Px) (II). The use of this test system made it possible to estimate the amount of a biologically active hormone and express it in concentration units.

Calibration curves were built for test systems I and II with the use of purified C-terminal rAMH fragment as the standard; it was obtained via proteolysis by the plasmin of a full-length hormone according to a previously described method [9] and its subsequent purification by affinity chromatography on ACMIS-1-sepharose immunosorbent.

Full sized rAMH was isolated by immunoaffinity chromatography from the culture fluid of the producer strain. Immunosorbents were prepared via immobilization of M2 and ACMIS-1 monoclonal antibodies on CNBr-activated sepharose FF (Pharmacia, Sweden) according to the manufacturer’s protocol. The hormone fragments were obtained from purified rAMH after prolonged storage in 20 mM phosphate buffer, pH 7.4, at 37°C. The rAMH preparations were analyzed via electrophoresis of proteins in a polyacrylamide gel (ESP-PAGE) in the presence of DDS-Na and/or in the absence of a reducing agent (β-mercaptoethanol) according to a previously described procedure [10]. In this case, a separating gel with a 4–20% gradient concentration of acrylamide and a concentrating gel with a 5% concentration were used. To conduct ESP-PAGE, 1–2 mg/mL of protein were incubated for 5 min at 95°C in the presence of β-mercaptoethanol (final concentration of 2%) or without it, and samples of 15 μL were then applied to the gel. ESP-PAGE was carried out in the BioRad MiniProtean Tetra Cell system (BioRad, USA) at a voltage of 40 V per gel for 20 min and further at a current of 200 mA until the end of the electrophoretic separation.

To visualize the results, the gel was stained with Coomassie G-250, followed by washing with a 5% solution of acetic acid at room temperature.

The rAMH fragments were fractionated by reverse phase high performance liquid chromatography (RP-HPLC) on an Agilent 1260 chromatograph (Agilent Technologies, Germany). Separation was performed on a C18 Jupiter Phenomenex column (4.6 × 250 mm, pore diameter 300 Å) (GE Healthcare, Sweden). Elution was carried out for 15 min, at a flow rate of 1.5 mL/min in a trifluoroacetic acid–acetonitrile gradient system: 20–70%. Components were detected during chromatography at 280 nm.

For the mass spectrometric study, derivatives of the C-terminal rAMH fragment were treated with dithiothreitol (DTT) (final concentration of 15 mM) and then with iodine acetamide (final concentration of 50 mM). The resulting polypeptides were again separated by RP-HPLC. The polypeptides were hydrolyzed with trypsin (Promega, United States) or with GluC protease (Sigma, United States) in 50 mM of ammonium bicarbonate solution for 5 or 24 hours, respectively, at 37°C according to the manufacturer’s instructions. The resulting peptides were mixed with an HCCA matrix (alpha-cyano-4-hydroxycinnamic acid, Bruker, Germany), applied to the GroundSteel target (Bruker, Germany), and air-dried.

Mass spectra were obtained on an UltrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker, Germany) in positive ion mode. Three thousand laser pulses were summed for each spectrum. Proteins were identified with the Mascot search algorithm (http:// matrixscience.com) with simultaneous accessing of the SwissProt database and the local database (to which the rAMH sequence was preliminarily included). The error was limited to 50 ppm; methionine oxidation and cysteine carbamidomethylation were also indicated as possible peptides modifications. The SequenceEditor program was also used to predict the peptides sequences by the ions detected in the mass spectra.

RESULTS AND DISCUSSION

A preliminary analysis of rAMH preparations obtained after the cultivation of CHO-MIS #26 producer cells showed that the population of hormone molecules accumulating in the culture medium is heterogeneous. It is represented not only by full-sized rAMH (rAMH*) but also by its derivatives (rAMH**), among which is a noncovalent complex of C- and N‑terminal fragments, AMG fragmented along the same chain, and a mixture of isolated C- and N-terminal fragments (Fig.1).

Fig. 1.

Schematic representation of the rAMH derivatives that accumulate in the culture fluid of the producer cells during cultivation.

The method of immunoaffinity chromatography with antibodies of various specificity was applied to separate the pAMP derivatives and isolate homogeneous fractions of the hormone. The method of two-step affinity chromatography on ACMIS-1-sepharose and M2-sepharose immunosorbents was used to isolate full-length rAMH. Columns containing immunosorbents were equilibrated with 20 mM phosphate buffer, pH 7.4, containing 0.15 M NaCl. At the first purification stage, clarified culture liquid was applied to an AFMIS-1-Sepharose column for the sorption of fragmented rAMH. The non-adsorbed components were applied to a column with M2-sepharose on which rAMH* was sorbed. After the column was washed with an equilibration buffer and a buffer containing 1 M NaCl, the rAMH* was eluted from the immunosorbent with a solution of 3 M MgCl2 in 20 mM phosphate buffer, pH 7.4.

Electrophoretic analysis of several eluates containing purified rAMH* showed that it migrated in the gel in the region of apparent molecular masses of 150 kDa in the form of a broad band, which is typical of glycosylated proteins, and it did not contain any impurity components (Fig. 2).

Fig. 2.

Electrophoresis in PAAG of three (1–3) rAMH* preparations (1 μg protein/lane) purified via two-step affinity chromatography on ASMIS-1 and M2-sepharose immunosorbents. M—protein markers of molecular masses.

To study the stability of purified rAMH*, its solution (1 mg/mL) was dialyzed against 20 mM phosphate buffer, pH 7.4, sterilized by microfiltration, and stored at 37°C. Every 5 days, samples were taken from it to determine the biological activity and the total amount of formed rAMH**. The latter was determined by ELISA with test systems I and II described above. The results are shown in Fig. 3. Purified rAMH*, which represents the parent drug, was not capable of binding to the receptor and, according to the results of the analysis in two test systems, was characterized by a zero level of fragmentation. Since it was incubated from 0 to 25 days, the rAMH** content in the samples increased, as evidenced by the data obtained with test system I (Fig. 3, curve 1). The coincidence of concentrations when tested by both test systems (I and II) indicated that all of the protein contained in these samples was capable of interacting with MISRII and, consequently, was biologically active. With further incubation from 25 to 45 days, the total amount of rAMH** in the samples continued to increase, while the proportion of biologically active substance reached the maximum by 30 days and then began to decrease (Fig. 3, curve 2).

Fig. 3.

Content of biologically active rAMH** detected after prolonged incubation of purified rAMH* in 20 mM phosphate buffer, pH 7.4, at 37°C with test system I (ACMIS-3–rAMH*–ACMIS-4-Ph (1) and test system II I4–MISRII + Fc–rAMH–ACMIS-4-Px (2).

According to the differential specificity of test system I, an increase in the concentration of recognizable material in incubation samples over time should be associated with rAMH fragmentation. This was clearly confirmed by the results of ESP-PAGE (Fig. 4). The presented electrophoregram showed that the amount of rAMH* (lane 1) significantly decreased during incubation for 25 days, up to an almost complete disappearance, while the number of fragmented heterogeneous components diffusely distributed in the gel increased. At the same time, a 20-kDa protein accumulated in the sample.

Fig. 4.

EP-PAAG of the initial rAMH* (1) and rAMH after incubation in 20 mM phosphate buffer, pH 7.4, for 15 (2) and 25 days (3) at 37°С. M—protein markers of molecular masses.

To identify the products of spontaneous rAMH* fragmentation, which are formed during incubation in 25 days (with the maximum receptor-binding activity of the protein, see Fig. 3), analysis by RP-HPLC was conducted (Fig. 5a). As can be seen in the chromatogram, the product was divided into two main components and two minor components, which are designated as 2 and 4 and 1 and 3, respectively.

Fig. 5.

RP-HPLC rAMH* after incubation in 20 mM phosphate buffer, pH 7.4, for 25 days (a) and the C-terminal fragment of rAMH obtained after the processing of full-length hormone with plasmin (b).

The obtained data suggested that the peak recorded by the retention time on the column in the range of 13–14 min and detected on the electrophoregram (Fig. 4, lane 3) as a 20-kDa protein corresponded to the rAMH C-terminal fragment, and peak 4 combined heterogeneous fragments that were not localized on the electrophoregram in the form of clear bands.

In a later work, a 20-kDa fragment associated (according to literature data) with the manifestation of AMH biological activity was studied in more detail [4]. In order to test and confirm the assumption that this spontaneously formed fragment belonged to the C-terminal rAMH homodimer, a standard sample of this derivative was obtained independently via rAMH* treatment with a plasmin (90 min, 37°C with a 1 : 25 plasmin : protein ratio) [10]; it was purified by immunoaffinity chromatography on ACMIS-1-sepharose immunosorbent and subjected to RP-HPLC (Fig. 5b). The retention time of the rAMH* C-terminal fragment formed after its treatment with plasmin exactly coincided with the retention time of Peak 2—the product of spontaneous fragmentation of rAMH* during incubation for 25 days (Figs. 5a, 5b). This confirmed its relation to the C-terminal fragment of the hormone. It could be also concluded that the spontaneous proteolysis of rAMH proceeded through the Arg–Ser bond, since it was shown that plasmin, like other enzymes responsible for cleaving rAMH to C- and N-terminal fragments, cleaves the polypeptide chain in vivo precisely at this site [8, 11]. The protein contained in peak 4 (Fig. 5a), according to the results of direct solid-phase ELISA with M2 and ACMIS-1 antibodies, was identified as a mixture of rAMH* fragmented by one chain and hormone fragments containing the N-terminal region.

The causes of rAMH inactivation during the incubation process were also studied. C-terminal fragments were isolated from biologically active rAMH** (25 days of incubation for preparation 1) and partially inactivated rAMH** (45 days of incubation for preparation 2) by RP-HPLC. Their ability to bind to the receptor in test system II was studied. It turned out that the nature of the binding of the C-terminal fragments contained in preparations 1 and 2 with the extracellular part of MISRII is different (Fig. 6). Thus, preparation 1 was more intensively bound to the receptor, and this binding was characterized by a tendency to saturate. These results indicated that the reason for the loss of the bind to the MISRII biological activity of rAMH** was caused by inactivation of the C-terminal fragment, which, according to the literature, makes the greatest contribution to the manifestation of the biological activity of the hormone [4].

Fig. 6.

Dependence of the intensity of binding of the rAMH** components with the extracellular part of the MISRII receptor, determined using test system II (I4– MISRll + Fc–rAMH–ACMIS-4-Px), from the incubation under the physiological conditions of full-length rAMH within 25 (1) and 45 days (2).

For a detailed study of the molecular structure of the partially inactivated C-terminal fragment (preparation 2), it was analyzed by RP-HPLC. The composition of this fragment turned out to be heterogeneous (Fig. 7). Components 1 and 2 were rechromatographed under the same conditions (Fig. 8). As a result of the separation, two homogeneous (according to the RP-HPLC data) C-terminal fragments were obtained and designated as C1 and C2. The study of their biological activity with the test system II indicated that C1 was inactive and C2 showed high activity. An additional study showed that the value of C2 with the MISRII association constant calculated by the Scatchard method was Kа = 4 ± 0.32 × 1010 nM–1.

Fig. 7.

RP-HPLC of the C-terminal fragment isolated after incubation of the rAMH* for 45 days.

Fig. 8.

RP-HPLC of C1 (1) and C2 (2) peptides isolated from partially inactivated C-terminal fragment.

The obtained C1 and C2, as well as the initial partially inactivated preparation of the C-terminal fragment representing their mixture, were analyzed by EP-PAGE (Fig. 9). After separation under nonreducing conditions (Fig. 9, lanes 13), their electrophoretic profiles turned out to be the same, while their molecular weights were about 20 kDa, which corresponded to the mass of the C-terminal hormone homodimer.

Fig. 9.

EP-PAAG in non-reducing (1–3) and reducing conditions (4–6) of C2 fragment (biologically active form, 1 and 4), C1 (biologically inactive form, 3 and 6), purified by fractionation by RP-HPLC, and the original rAMH* C-terminal homodimer, which is formed after incubation of the rAMH in physiological conditions for 45 days at 37°C (2 and 5); M – protein markers of molecular masses.

However, the results of electrophoretic analysis under reducing conditions indicated that the studied C1 and C2 differed significantly. The preparation obtained from rAMH* after 45 days of incubation (Fig. 9, lane 5) representing a mixture of C1 and C2 contained two components, the apparent molecular weights of which were about 12.5 and 6 kDa. It should be noted that the biologically active C-terminal fragment of pAMH–C2 (lane 4) migrated as one component with a molecular weight of about 12.5 kDa, which corresponded to the molecular weight of the C-terminal homodimer polypeptide chain. Component C1 (lane 6) also contained an individual polypeptide, but it was characterized by almost half of the molecular weight of about 6 kDa, which indicated the proteolytic cleavage of both polypeptide chains of the rAMH C-terminal homodimer.

The results indicated that prolonged rAMH incubation resulted in its fragmentation with the formation of a biologically active C-terminal fragment, which was further subjected to proteolytic processing and split into 6 kDa fragments. At the same time, all of them were linked in a single complex with a molecular mass of about 20 kDa due to the presence of numerous intramolecular disulfide bonds in the C-terminal fragment of the rAMH molecule [12].

For a more detailed analysis of the molecular structure of the fully inactivated C1 protein fragments and the determination of the proteolysis site by which the rAMH-terminal dimer was inactivated, it was treated with DTT in the presence of 4 M guanidine hydrochloride (solution in 100 mM Tris-HCl buffer, pH 8.0) and then by iodine acetamide to block the reverse closure of disulfide bonds. The analysis was carried out by RP-HPLC (Fig. 10). The main major fractions (peaks 1 and 2) were then studied by MALDI mass spectrometry.

Fig. 10.

RP-HPLC of fragments (1 and 2) of C1 peptide after reduction by DTT and subsequent modification with iodine acetamide.

Analysis of the MALDI mass spectra obtained as a result of the study of the two peptides described above made it possible to establish that the fragmentation of the C-terminal rAMH dimer proceeded in its monomers in the leucine motif after the first leucine (58th amino acid residue) (Fig. 11). Peptides that indicate a break in the polypeptide chain in residues that are not characteristic of the specificity of the action of the two used proteases were identified in the spectra of a 6 kDa fragment. Thus, an ion with m/z = 801.428 was found after treatment with trypsin; it corresponded to the peptide YGNHVVL and the protease GluC (m/z = 3290.497), which corresponded to the peptide TYQANNCQGVCWPQSDRNPRYGNHVVL (Fig. 12). In this case, the ions corresponding to nonfragmented peptides (in particular, in the case of trypsin—YGNHVVLLLK) were absent in the spectra.

Fig. 11.

Site of proteolysis in the amino acid sequence of the monomer of the C-terminal fragment of rAMH during specific fragmentation of the hormone.

Fig. 12.

The region of the characteristic ion with m/z = 801.428 (a) and m/z = 3290.497 (b) MALDI mass spectra of one of the two fragments of the rAMH C-terminal dimer formed during specific fragmentation of the complete hormone chain; the amino acid sequences of the peptides corresponding to these ions are shown.

Thus, on the basis of the above results, it can be concluded that the prolonged incubation of affinity purified, full-length rAMH at a pH of 7.4 resulted in spontaneous fragmentation of the protein by the peptide bond between the Arg and Ser amino acid residues and the accumulation of the functionally active C-terminal fragment of rAMH. During further incubation, the C-terminal fragment was cleaved in a bond between two leucine residues (at positions 58 and 59) located in the central part of the monomer, and it passed into an inactive form. Such cleavage was presumably observed in highly purified rAMH without the participation of extraneous enzymes and could be due to the presence of its own proteolytic activity in this protein.

Thus, it was shown that in vitro under physiological conditions that the spontaneous processing of full-length rAMH proceeded with the formation of N- and C-terminal homodimers and that the biological activity of the latter was established. In the control experiment, the C-terminal fragment of rAMH was obtained via limited proteolysis of purified full-length rAMH with plasmin. It is established that the C-terminal fragment of rAMH resulting from spontaneous hydrolysis is identical to the C-terminal homodimer obtained by the splitting of a full-sized hormone molecule with plasmin. It was shown that prolonged incubation of the C-terminal homodimer (activated by rAMH) under physiological conditions led to its additional cleavage at the site between the 58th and 59th amino acid residues of leucine (Leu-Leu) and the loss of the ability to bind to the specific rAMH receptor, type II MISRII.

Notes

COMPLIANCE WITH ETHICAL STANDARDS

The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.

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Copyright information

© Pleiades Publishing, Inc. 2019

Authors and Affiliations

  • A. Ya. Rak
    • 1
    • 2
    Email author
  • A. V. Trofimov
    • 1
  • E. A. Protasov
    • 1
  • S. V. Rodin
    • 1
  • A. V. Zhahov
    • 1
  • Ya. A. Zabrodskaya
    • 1
    • 3
  • A. M. Ischenko
    • 1
  1. 1.State Research Institute of Highly Pure Biopreparations, Federal Medical-Biological Agency (FMBA)St. PetersburgRussia
  2. 2.St. Petersburg State UniversitySt. PetersburgRussia
  3. 3.Research Institute of Influenza, Ministry of Health of RussiaSt. PetersburgRussia

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