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Endocrinology Vol. 139, No. 3 999-1005
Copyright © 1998 by The Endocrine Society

ARTICLES

The Greater Glycan Content of Recombinant Human Thyroid Peroxidase of Mammalian Than of Insect Cell Origin Facilitates Purification to Homogeneity of Enzymatically Protein Remaining Soluble at High Concentration1

Jin Guo, Sandra M. McLachlan, Scott Hutchison and Basil Rapoport

Thyroid Molecular Biology Unit, Veterans Administration Medical Center and University of California (J.G., S.M.M., B.R.), San Francisco, California 94121; and Nichols Institute Diagnostics (S.H.), San Juan Capistrano, California 92690

Address all correspondence and requests for reprints to: Dr. Basil Rapoport, M.D., Thyroid Molecular Biology Unit (111T), VeteransAdministration Medical Center, 4150 Clement Street, San Francisco, California 94121.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Structural studies on thyroid peroxidase (TPO), a major thyroid autoantigen, require milligram amounts of pure protein. We found that the human TPO ectodomain (amino acid residues 1–848) generated in insect cells did not remain in solution at high concentrations after affinity purification. In contrast, the TPO ectodomain secreted by mammalian (Chinese hamster ovary) cells, although generated to a lesser extent (1 vs. 8 mg/liter), remained in solution at high concentration (10 mg/ml) after purification to homogeneity. This purified material was well recognized by TPO autoantibodies, but lacked enzymatic activity. We attempted to restore activity by culturing the Chinese hamster ovary cells in the presence of added heme. TPO enzymatic activity was clearly detected in conditioned medium from cells cultured in hematin and hemin, but not in protoporphyrin IX (all at 1 mg/liter). Heme prosthetic group incorporation into affinity-purified TPO was highest for hematin and hemin, but unchanged for protoporphyrin IX (OD 410/280 nm ratios of 0.25, 0.23, and 0.14, respectively). Enzymatic activity was now evident with hemin (mean ± SE, 27.2 ± 2.6; n = 3; guaiacol units/mg protein), hematin (24.1 ± 1.6), and, to a lesser extent, protoporphyrin IX (3.6 ± 0.2). Culturing cells in 20 mg/liter hematin, the maximum concentration tolerated, increased enzymatic activity even further (45.6 ± 0.6 guaiacol units/mg protein). All purified TPO preparations were homogeneous on PAGE and of similar size (105 kDa). Enzymatic deglycosylation showed a complex carbohydrate contribution of 13 kDa (unlike the 2.3 kDa in insect cell TPO).

In conclusion, this is the first report on the purification to homogeneity of recombinant human TPO of mammalian cell origin. Unlike TPO generated in insect cells, mammalian TPO remains soluble at high concentration, possibly because of its greater carbohydrate content. This enzymatically active, recombinant human TPO may be useful for future structural studies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
HUMAN thyroid peroxidase (hTPO), the primary enzyme in multiple stages of thyroid hormone biosynthesis (reviewed in Ref.1), is also the major thyroid autoantigen to which autoantibodies are directed in patients with thyroid autoimmunity (reviewed in Ref.2). To understand both of these important functions of TPO, a glycoprotein with a heme prosthetic group, it will be necessary to determine its three-dimensional structure. For this purpose, milligram amounts of native, enzymatically active, and immunologically functional hTPO protein are required. Very recently, 20 mg hTPO were purified from Graves’ thyroid tissue, but the crystals generated were unsuitable for x-ray diffraction analysis (3). Because kilogram quantities of human thyroid tissue are rarely available, recent efforts have focused on generating the recombinant protein, made feasible by the cloning of a complementary DNA (cDNA) clone for porcine TPO in 1986 (4) and the full-length porcine (5) and human (6, 7, 8) TPO cDNA sequences in 1987.

In the subsequent decade, numerous groups, including our own, have reported the generation of large amounts of TPO, either the full-length molecule (9, 10) or its extracellular domain (11, 12), in insect cells using the baculovirus system. Indeed, it is the present perception that "only ... the insect cell/baculovirus system is capable of yielding milligram quantities of recombinant, native enzyme" (3). This perception is correct only to the extent that we (and, perhaps others) have not previously reported the purification to homogeneity of milligram amounts of recombinant TPO from mammalian cells. Moreover, we observed important differences between recombinant TPO of insect cell and that of mammalian cell origin that may be of importance in obtaining TPO crystals of sufficient quality for x-ray diffraction studies.

The present report, therefore, describes our experience over the past 7 yr with the purification of large quantities of hTPO in both insect cells and in mammalian [Chinese hamster ovary (CHO)] cells. In both instances, to facilitate purification, we expressed a recombinant TPO molecule with a stop codon introduced at the junction of the ectodomain and the plasma membrane, a maneuver that we had previously found converted the membrane-associated protein of 933 amino acids (including signal peptide) into an 848-residue molecule secreted into the culture medium (13).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Affinity purification of recombinant hTPO ectodomain
Expression of the recombinant human TPO ectodomain by CHO (13) and insect (Sf9) cells (11) has been described previously in detail. In the case of the mammalian cells, the TPO cDNA transgenome was amplified using a dihydrofolate reductase minigene (14). In some instances the culture medium of the CHO cells was supplemented with the indicated concentrations of hemin, hematin, or protoporphyrin IX (all from Sigma Chemical Co., St. Louis, MO). Conditioned medium (2–3 days of culture for both CHO and Sf9 cells, stored at -80 C) was thawed slowly, filtered (0.22 µm), and applied (0.5–3 liters at 1.5 ml/min at room temperature) to a column with 15 ml Sepharose-linked mouse monoclonal antibody to hTPO. The monoclonal antibody, generated by one of us (S.H., Nichols Institute, San Juan Capistrano, CA) by immunizing mice with recombinant, secreted human TPO, was an IgG1 {kappa}. After extensive washing with PBS, pH 7.4, the protein was eluted (1.5 ml/min) with 0.2 M glycine, 0.15 M sodium chloride, and 0.02% sodium azide, pH 2.3. Fractions (2 ml) were immediately neutralized with 0.4 ml Tris, pH 8.0. TPO typically eluted from the column in fractions 8–15. Protein concentrations in the fractions were measured by spectrophotometry at OD 280 nm (extinction coefficient of 1% TPO, 1-cm path of 17.9). This extinction coefficient was based on a direct estimate of the mass of purified protein by Bradford analysis (15) as well as by Coomassie blue staining of pure protein electrophoresed on polyacrylamide gels relative to known protein standards included in parallel lanes. Fractions with an OD greater than 0.1 were pooled; dialyzed against 10 mM HEPES or Tris, pH 7.4, and 0.02% sodium azide; and concentrated with a Centriprep 30 (Amicon, Beverly, MA). In preparations in which enzymatic activity was to be determined, azide was omitted from the dialysis buffer. Incorporation of the heme prosthetic group was estimated by the OD 410/280 nm ratio. TPO purity was assessed by PAGE (7.5% or 10%) in the presence of SDS and 0.7 M ß-mercaptoethanol, followed by Coomassie blue staining.

Gel filtration of TPO and TPO-Fab complexes
Purified mammalian cell TPO (100 µg), an equimolar amount (50 µg) of purified TPO autoantibody Fab TR1.9 (16), or a mixture of these two proteins (preincubated for 3 h at room temperature) was applied (1 ml) to a Sephacryl S300 (Pharmacia, Piscataway, NJ) column (70 x 1.6 cm) equilibrated with 20 mM Tris, pH 7.0, and 150 mM NaCl. The column was developed with the same buffer (1 ml/min), and the OD at 280 nm was measured in 1-ml fractions.

TPO enzymatic activity
TPO enzymatic activity in conditioned medium or in the purified protein was determined using the guaiacol assay. Before assay, samples were dialyzed against calcium- and magnesium-free PBS. The reaction mixture (1-ml total volume) in the same buffer contained 33 mM guaiacol (Sigma), 0.5 mg/ml BSA, and the sample to be assayed (typically 5–10 µg protein). The reaction was begun by the addition of 5 µl 0.6 M H2O2, and OD was monitored at 470 nm. Guaiacol units were defined as {Delta}OD 1.0/min (17, 18).

Enzymatic deglycosylation
Purified TPO preparations were digested with endoglycosidase F and endoglycosidase H according to the protocol of the manufacturer (New England Biolabs, Beverly, MA). In brief, samples (~5 µg) were denatured for 15 min at 95 C in 0.5% SDS and 1% ß-mercaptoethanol. Endoglycosidase F digestion (1000 U, 4–16 h at 37 C) was performed in 50 mM Na phosphate, pH 7.5, and 1% Nonidet P-40. Endoglycosidase H digestion (1000 U, 4–16 h at 37 C) was in 50 mM Na citrate, pH 5.5. Reaction volumes were 80 µl. For O-glycosidase digestion, 10 µg TPO were first treated with neuraminidase from Clostridium perfringens type X (Sigma) in 50 µl 50 mM phosphate buffer, pH 6.4 (1.5 mU, 3.5 h at 37 C). O-Glycosidase (1 mU; Boehringer Mannheim, Indianapolis, IN) was added, and the incubation was continued overnight at 37 C, after which endoglycosidase F digestion was performed as described above. Aliquots of all digestions in Laemmli buffer (19) were subjected to SDS-PAGE (7.5%) under reducing conditions followed by staining with Coomassie blue.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Purification of recombinant TPO from insect cells
As mentioned above, four groups have reported the expression of human TPO, either the whole molecule (9, 10) or the ectodomain (11, 12), in insect cells using a baculovirus vector. All preparations have been found to be very effective in terms of their recognition by autoantibodies in the sera of patients with autoimmune thyroid disease. Full-length TPO has been purified by reverse phase HPLC (10). However, the small yield and elution under stringent solvent conditions (~50% acetonitrile and 0.1% trifluoroacetic acid) would make crystallographic studies difficult. Very recently, sequential ion exchange chromatography and gel filtration were used to obtain a TPO ectodomain of relatively high purity (12). However, it remains to be seen whether crystallization would be impeded by the remaining contaminating proteins.

Four years ago, we succeeded in purifying to homogeneity the TPO ectodomain generated in insect cells by affinity chromatography using a Sepharose-linked monoclonal antibody to TPO (see below). However, we encountered a major problem with insect cell TPO in that it did not remain in solution at high concentrations and was, therefore, unsuitable for crystallographic studies. A possible reason for the relative insolubility of insect cell TPO is presented below.

Purification of recombinant TPO from mammalian cells
The lower level of expression of recombinant proteins in mammalian cells compared with that in insect cells is a major handicap to obtaining milligram quantities of purified material. Thus, TPO yields of 8.5–30 mg/liter have been reported for insect cells (11, 12). In contrast, even after transgenome amplification in CHO cells of a cDNA for a form of TPO engineered to be secreted rather than being membrane bound (13, 14), the concentration of TPO secreted into culture medium only attained 1 mg/liter. Although time-consuming and expensive, in view of our experience with insect cell TPO, we returned to the purification of recombinant TPO from mammalian cells. The soluble human TPO secreted by these cells was readily purified to homogeneity by a single passage over a Sepharose-anti-TPO affinity column. Thus, the recombinant TPO migrated as a single band of ~105 kDa on PAGE (Fig. 1Go). Most important, unlike the insect cell material, the TPO generated by mammalian cells remained in solution after affinity purification, even at concentrations as high as 10 mg/ml.



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  		Figure 1. Affinity-purified soluble human TPO secreted by CHO cells. Material was subjected to SDS-PAGE (10%) under reducing conditions followed by staining with Coomassie blue.

 
The purified, mammalian TPO is immunologically intact in terms of its recognition by TPO autoantibodies in patients’ sera. Indeed, it is this material that we radiolabeled and used in numerous studies on the molecular cloning and characterization of human monoclonal autoantibodies to TPO (reviewed in Refs. 20 and 21). We further examined the interaction between unlabeled TPO with purified, recombinant TPO autoantibodies expressed as Fab. To confirm the immunological integrity of purified, unlabeled TPO, we preincubated the TPO with an equimolar concentration of a TPO-specific Fab. As determined by gel filtration, the entire recombinant TPO peak shifted to the left, consistent with all of the material forming a TPO-Fab complex of higher mol wt (Fig. 2Go).



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  		Figure 2. Interaction between purified, recombinant mammalian TPO with a purified, recombinant TPO autoantibody expressed as Fab. TPO (0.1 mg) was preincubated with a TPO autoantibody Fab (TR1.9; 0.05 mg; see Materials and Methods) (16). One-milliliter aliquots of TPO alone, Fab alone, or TPO plus Fab complex were applied to a Sephacryl S-300 column. Fractions are 1 ml.

 
Enzymatic activity of recombinant TPO
Previously, in our studies on recombinant TPO generated in CHO cells, we had found low level enzymatic activity in unpurified membrane-associated enzyme (22) as well as in culture medium containing the secreted TPO ectodomain (13). However, the affinity-purified TPO ectodomain of both insect cell (11) and mammalian cell origin was devoid of enzymatic activity. Because of previous reports that hematin can enhance TPO enzymatic activity in extracts of human thyroid (23, 24), we attempted restoration of TPO enzymatic activity in recombinant TPO ectodomain secreted by mammalian (CHO) cells. Spectroscopically, a small amount of an unknown form of heme prosthetic group was present in the purified, inactive enzyme, with an OD 410/280 nm ratio of 0.14–0.18 in different preparations (Table 1Go). These values were considerably lower than those reported for TPO purified from thyroid tissue, i.e. 0.27–0.65 (18, 25, 26, 27). Because the exact nature of the heme present in thyroidal TPO in thyrocytes is unknown, we cultured the CHO cells in medium containing three different types of heme: hematin, hemin, and protoporphyrin IX. The latter compound does not contain iron and can be regarded as a negative control. Medium from CHO cells cultured with hemin and hematin, but not with protoporphyrin IX (all at 1 mg/liter), contained low level, but clearly detectable TPO enzymatic activity (data not shown). Consistent with these values, after affinity purification of TPO from large volumes (500 ml) of conditioned medium, the OD 410/280 nm ratios were markedly increased with hemin and hematin, but not with protoporphyrin IX (Table 1Go). To attain an even higher ratio, we cultured cells at the maximum tolerated concentration of the heme compound that gave the highest ratio (20 mg/liter hematin). Indeed, the OD 410/280 nm ratio in TPO purified from this medium of 0.75 was higher than that attained for nonrecombinant, thyroidal TPO (27). The purity of all large scale TPO preparations was confirmed by PAGE (Fig. 3Go).


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  		Table 1. Enzymatic activity of purified TPO

 


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  		Figure 3. Purity of TPO from mammalian cells incubated in medium containing different heme prosthetic groups. CHO cells overexpressing the TPO ectodomain cDNA were cultured in the presence of the indicated concentrations of protoporphyrin IX, hemin, and hematin. TPO was affinity purified from conditioned medium (0.5 liters; see Materials and Methods) and subjected to SDS-PAGE (10%) under reducing conditions followed by staining with Coomassie blue.

 
Enzymatic activities of purified TPO were markedly enhanced when the CHO cells were cultured in hemin and hematin (1 mg/liter; Table 1Go and Fig. 4Go). As anticipated, essentially no activity was detected in TPO purified from the medium of CHO cells cultured in a similar concentration of protoporphyrin IX. TPO purified from cells cultured in a 20-fold higher concentration of hematin (20 mg/liter) had enzymatic activity approximately 2-fold greater, suggesting that this level of activity was the maximal that could be attained in this recombinant material (Table 1Go and Fig. 4Go). During the course of these studies, other investigators studying TPO holoenzyme (10) and ectodomain (12) generated in insect cells found a similar lack of enzymatic activity. Such activity could be restored in the holoenzyme and ectodomain material by including either 1 mg/liter hematin (10) or the heme biosynthesis precursor {delta}-aminolevulinic acid in the insect cell culture medium (12). However, our observation that hemin restored enzymatic activity in TPO secreted by CHO cells contrasts with the ineffectiveness of this compound with insect cell TPO (12).



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  		Figure 4. Enzymatic activity of purified, recombinant mammalian TPO ectodomain preparations. Material was affinity purified from conditioned medium of the same CHO cell line cultured in the presence of the indicated types and concentrations of heme. The guaiacol assay was performed as described in Materials and Methods, with about 10 µg TPO protein.

 
Because of the different units used to express TPO enzymatic activity in the literature, it is difficult to relate the specific activities of the different forms of recombinant TPO. There are at least three different definitions of enzymatic activity in the guaiacol assay (Table 1Go). To compare the specific activities of purified recombinant and thyroidal TPO, we normalized the activity values reported in different studies to a guaiacol unit being equivalent to an increase in OD (470 nm) of 1.0/min. On this basis, the specific activity of our most active recombinant TPO of mammalian cell origin (46 U/mg protein) was quite similar to that of recombinant TPO generated in insect cells (12) as well of thyroidal human TPO purified and assayed in the same laboratory (12). We cannot explain why these TPO-specific activities (both recombinant and natural) are far lower than those reported previously by Ohtaki et al. (27) and Taurog et al. (26). Neither 1) affinity purification vs. conventional purification, 2) pig vs. human TPO, 3) recombinant vs. thyroidal TPO, 4) TPO solubilized from thyroid membranes using detergent and trypsin (~90 kDa) (18, 26, 27, 28, 29) vs. TPO ectodomain secreted into the medium (~105 kDa), nor 5) absolute heme prosthetic group content is a viable explanations (Table 1Go).

The most plausible explanation for the low specific activity of the recombinant TPO in the present study is that the heme prosthetic group is not covalently bound as is the case in myeloperoxidase (30) and lactoperoxidase (31, 32, 33). Thus, culturing CHO cells in hematin or hemin might not lead to efficient incorporation of the heme prosthetic group. The very high value of 0.75 (OD 410/280 nm) obtained in the presence of a saturating hematin solution in association with the relatively low enzymatic activity further suggests that the heme is not physiologically incorporated into the TPO protein. It should be noted that, at least for TPO generated in eukaryotic insect cells, the inclusion of a heme precursor ({delta}-aminolevulinic acid) in the culture medium produced TPO with a specific enzymatic activity (12) (Table 1Go) similar to that of the material generated in CHO cells. It should also be pointed out that CHO cells are, at least under some conditions, capable of synthesizing and covalently binding a heme prosthetic group, as demonstrated for myeloperoxidase (34). We are unable to explain why the recombinant MPO in this study, unlike recombinant mammalian TPO produced in the same cell line, compared very favorably to naturally isolated MPO in terms of specific activity and prosthetic group incorporation.

Glycan moieties on human TPO
Although it is well established that TPO is a glycoprotein (18), the glycan component does not contribute to TPO recognition by autoantibodies (35, 36, 37) or to its enzymatic activity (36, 38). However, the poor solubility at high concentration of TPO ectodomain secreted by insect cells relative to similar material of mammalian cell origin (see above) as well as our observations on the recombinant TSH receptor expressed in the same two systems (39, 40) led us to compare the glycan moieties on TPO secreted by insect cells and mammalian cells.

The purified TPO ectodomain generated by CHO cells was enzymatically deglycosylated by endoglycosidase F, but not by endoglycosidase H, indicating that it contains complex, rather than high mannose, carbohydrate (Fig. 5AGo). After deglycosylation, the polypeptide backbone of the TPO ectodomain was approximately 92 kDa, consistent with the size predicted from its amino acid composition and suggesting a glycan contribution of about 13 kDa. The glycan properties of TPO purified from cells cultured in 20 mg/liter hematin were identical to those of the enzyme generated in nonsupplemented medium (Fig. 5AGo). Of interest, the complex carbohydrate on the secreted TPO ectodomain differs from the high mannose carbohydrate (endoglycosidase H sensitivity) previously observed with the TPO holoenzyme expressed in CHO cells (35). This difference may be explained by the fact that the holoenzyme was detected in metabolically labeled whole cell extracts that contain large amounts of intracellular precursors. On the other hand, TPO secreted into the culture medium must be a mature, fully glycosylated product. After combined neuraminidase and O-glycosidase treatment, the mobility of the TPO ectodomain was, possibly, slightly greater on an extended electrophoretic run than that after endoglycosidase F alone (Fig. 5BGo), suggesting the presence of either no O-linked carbohydrate or a minimal amount of this material. The rationale for also removing N-linked glycans in these experiments was to maximize any change in migration that might be evident after O-glycosidase treatment.



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  		Figure 5. Enzymatic deglycosylation of TPO ectodomain generated by CHO cells. A, Endoglycosidase H (Endo H) or endoglycosidase F (Endo F) digestion (see Materials and Methods) of TPO purified from cells cultured in the absence or presence of 20 mg/liter hematin. B, O-Glycosidase digestion of the same material. Neuraminidase pretreatment facilitates the action of O-glycosidase. To maximize any change in migration that might be evident after O-glycosidase treatment, N-linked glycans were then removed by endoglycosidase F digestion. Material was subjected to SDS-PAGE (7.5%) under reducing conditions, followed by staining with Coomassie blue.

 
Relative to the mammalian cell material, the TPO ectodomain generated by insect cells was poorly glycosylated and heterogeneous. Thus, most of the molecules contained only approximately 2 kDa carbohydrate, as evident by deglycosylation with endoglycosidase F (Fig. 6Go), in agreement with a recent report (12). A smaller amount of more fully glycosylated material, of similar size to that generated by CHO cells, was also evident. As with mammalian cell TPO ectodomain, all insect cell material contained complex carbohydrate (endoglycosidase H resistant). Previous studies on insect cell TPO involving differential lectin reactivity have provided inconclusive results (12). The sugar composition of TPO holoenzyme from insect cells suggested the presence of N-linked high mannose as well as O-linked carbohydrate (10); however, it is difficult to relate these data to those obtained in the present study involving differential enzymatic deglycosylation of TPO ectodomain material. The most important conclusion from this somewhat confusing literature is that, regardless of the type of carbohydrate present on TPO of insect cell origin, the glycan content is very low relative to TPO expressed by mammalian cells.



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  		Figure 6. The TPO ectodomain generated by insect (Sf9) cells contains less carbohydrate than does TPO generated by mammalian (CHO) cells. Affinity-purified TPO ectodomain from cultures of CHO cells and Sf9 cells was enzymatically deglycosylated with endoglycosidase H (Endo H) or endoglycosidase F (Endo F; see Materials and Methods). Material was subjected to SDS-PAGE (7.5%) under reducing conditions, followed by staining with Coomassie blue.

 
In conclusion, this is the first report on the purification to homogeneity of recombinant human TPO of mammalian cell origin. Unlike similar material generated in insect cells, this mammalian TPO remains soluble at high concentration, possibly because of its greater carbohydrate content. This enzymatically active, recombinant human TPO will be useful for future structural studies.


   Footnotes
 
1 This work was supported by NIH Grant DK-36182. Back

Received August 22, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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