ARTICLE

Cytoplasmic Carbonic Anhydrase II of Rat Coagulating Gland Is Secreted via the Apocrine Export Mode

Correspondence to: Jürgen Seitz, Dept. of Anatomy and Cell Biology, Robert-Koch-Str. 6, 35037 Marburg, Germany.

	Summary

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Two different pathways for protein secretion are described for epithelial cells of rat coagulating gland and dorsal prostate: the classical merocrine and the alternative apocrine release mode. Apocrine-secreted proteins are synthesized on cytoplasmic polyribosomes and are subsequently exported in protrusions on the apical cell surface (aposomes). In this article we report the identification and purification to homogeneity of a 29-kD protein from the secretion of rat coagulating gland. N-terminal amino acid sequence analyses revealed 100% identity to rat brain carbonic anhydrase II (CAH II). In addition, the 29-kD protein showed CAH enzyme activity. On Western blot analysis, a polyclonal anti-CAH II antibody raised in rabbit reacted specifically with the rat and human but not bovine CAH II isoforms. Immunohistochemical studies on rat coagulating gland showed strong labeling for CAH II protein in aposomes. Immunoelectron microscopy confined CAH II protein to the cytoplasm and aposomes, whereas no staining was visible in the compartments of the classical merocrine route, the endoplasmic reticulum and Golgi apparatus. The resident cytoplasmic protein lactate dehydrogenase, however, was not found in the secretion. Taken together, the morphological and biochemical data clearly indicate that cytoplasmic CAH II from rat coagulating gland is specifically selected and then secreted via the apocrine pathway. (J Histochem Cytochem 46:505–511, 1998)

Key Words: secretion, carbonic anhydrase II, carbonate dehydratase II, apocrine, aposome, coagulating gland

	Introduction

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Carbonic anhydrases (CAH, carbonate dehydratase, EC 4.2.1.1) are a family of enzymes catalyzing the reversible hydration of carbon dioxide and thereby regulating the electrolyte and the acid–base balance in various organs. In addition to this physiological function, CAH hydrolyzes different esters, such as p-nitrophenylacetate, in vitro. This unspecific esterase activity can be used for CAH quantitation and for CAH detection in histochemical studies. To date, six major isoforms of CAH have been described in mammals: CAH I, CAH II, and CAH III are nonsecretory proteins localized in the cytoplasm, CAH IV is a plasma membrane protein, and CAH V is found in mitochondria. The only secretory isoform described is CAH VI from submandibular gland, which is exported by the classical merocrine mode (for overview see Dodgson 1991 ).

Immunohistochemical studies showed that CAH II isoforms were localized in the gastrointestinal tract, renal tubule, salivary glands, and in the male reproductive tract (Spicer et al. 1982 ; Harkonen and Vaananen 1988 ; Kaunisto et al. 1990 ; Harkonen et al. 1991 ). However, erythrocytes are the major source for CAH II, from which it has been purified to homogeneity (for review see Bergenhem 1996 ).

Functionally, the cytoplasmic CAH II isoform acts in concert with integral plasma membrane transport proteins to regulate bicarbonate and proton passage (Tashian et al. 1983 ). The first speculations that this enzyme can also be secreted came from immunohistochemical observations of Spicer at al. (1982) and Henningar et al. (1983) in salivary glands. Kaunisto et al. 1990 raised the possibility that CAH II may be exported from human accessory sex glands by an apocrine secretion mode.

Apocrine secretion, in contrast to the well-investigated merocrine pathway, does not include the endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles. Lacking a signal sequence for ER import, apocrine proteins are synthesized in the cytoplasm and are directly translocated into protrusions at the apical surface of epithelial cells, the so-called "blebs" or "aposomes" (Hawkins and Geuze 1977 ). Occasionally, aposomes originate from apical microvilli (Aumuller and Adler 1979 ). Transglutaminase (TGase; 65 kD), isolated from coagulating gland and dorsal prostate secretion, was the first protein demonstrated to follow this alternative export pathway (Seitz et al. 1990 ; Steinhoff et al. 1994 ). Recently, Manin et al. 1995 postulated a similar secretion mode for a protein from mouse vas deferens (MVDP) that is also lacking a signal peptide.

In this article we describe the characterization of a 29-kD protein from rat coagulating gland (synonym, anterior prostate; located adjacent to the concave curvature of the seminal vesicle in rodents) as a further apocrine-secreted protein and its identification as a CAH II isoform.

	Materials and Methods

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Animals
Animals were maintained under defined housing conditions in a constant day and night rhythm. All experiments were conducted in accordance with the German Law for the Use and Care of Laboratory Animals and were approved by the local animal experimentation review committee.

Purification of 29-kD Protein (CAH II) from Rat Coagulating Gland Secretion
Sexually mature male Wistar rats (290–310 g body weight; Charles River Wiga, Sulzfeld, Germany) were anesthetized and subsequently sacrificed by cervical dislocation. After removal of the coagulating glands, the intraluminal secretion was trickled into PBS containing 5 mM EDTA. After centrifugation of the secretion (15,000 x g, 20 min, 4C) and fractionated ammonium sulfate precipitation (30–51% saturation), proteins in the supernatant were separated by size exclusion chromatography on Superdex 200 (Pharmacia; Freiburg, Germany) using 50 mM Tris-HCl buffer, 5 mM EDTA (pH 8.0) for elution. Fractions containing the 29-kD protein were further enriched using anion exchange chromatography on Fractogel EMD DEAE-650 (s) (Merck; Darmstadt, Germany) equilibrated with 20 mM piperazine (pH 9.5). Bound proteins were eluted using a linear gradient from 0 to 1 M NaCl in 20 mM piperazine (pH 9.5). The Bio-Rad protein assay (Bio-Rad Laboratories; Richmond, CA) was used to determine protein concentrations. SDS-PAGE was performed according to Laemmli 1970 to check the purity of the fractions, using 4% stacking and 13% separating slab gels. Subsequently, the gels were stained with Coomassie brilliant blue R-250.

Amino Acid Sequence Analyses
In situ digestion was performed according to the method of Eckerskorn and Lottspeich 1989 separating 12 µg of 29-kD protein on a 13% SDS-PAGE. Subsequently, endoproteinase Lys-C of sequencing grade (1:10; w/w; Boehringer, Mannheim, Germany) for proteolytic cleavage and a Superspher 60 RP select B column (Merck) for peptide separation were used. Alternatively, 500 µg of rat coagulating gland 29-kD protein was reduced and carboxymethylated according to Allen 1981 . Fragmentation with trypsin and amino terminal sequence analyses were performed according to Linder et al. 1994 . For separation of tryptic peptides a Lichrospher column (WP 300 detection at 206 nm, 5 µm; Merck) was utilized.

Activity Assays
To determine CO₂ hydration activity, the 29-kD protein from rat coagulating gland was mixed with 20 mM Tris-HCl buffer (pH 8.3) at 4C. After addition of cold CO₂-saturated water, the elapsed time period required for the pH drop was recorded. CAH activity was determined in so-called Wilbur–Anderson units. Bovine CAH II was used as a positive control. Unspecific CAH esterase activity was assayed according to Wang 1981 . Positive controls were human CAH isoform I and II as well as bovine CAH II (Sigma; Munich, Germany).

Immunization of Rabbits with Rat 29-kD Protein (CAH II)
Fractions enriched with rat 29-kD protein (500 µg in total) were separated by 13% SDS-PAGE. The 29-kD band was excised, emulsified in Gerbu adjuvant 100 (Gerbu; Gailberg, Germany), and used for immunization of female New Zealand White rabbits (3 kg; Charles River). Antiserum (anti-CAH II) was finally obtained after three booster injections (4–6 week intervals).

Western Blotting
Western blotting was performed as described by Steinhoff et al. 1994 . Therefore, purified 29-kD protein, rat coagulating gland secretion, and coagulating gland cytosol, commercially available CAHs (Sigma), as well as LDH from hog muscle (Boehringer) were transferred onto nitrocellulose membranes and detected using either polyclonal anti-CAH II (1:300) as primary antibody or polyclonal peroxidase-conjugated antibody against rabbit muscle LDH (1:1000; Biotrend, Cologne, Germany). The peroxidase reaction was visualized with DAB/H₂O₂ as substrates.

Immunohistochemistry and Immunoelectron Microscopy
Small coagulating gland tissue fragments were immersion-fixed overnight in a mixture of 2.5% glutaraldehyde, 2.5% paraformaldehyde, and 0.05% picric acid in 100 mM cacodylate buffer (pH 7.3). Immunohistochemistry on semithin sections and immunoelectron microscopy on ultrathin sections were performed as described previously by Steinhoff et al. 1994 . Anti-CAH II antibody (1:50) was used as the primary antibody in the immunohistochemical experiments and was visualized either with DAB/H₂O₂ or Cy3 fluorescence-labeled secondary antibody. In immunoelectron microscopic studies, affinity-purified CAH II antibody (1:5) was used according to the method of Meinhardt et al. 1996 . The specimens were decorated with 20-nm colloidal gold-coated anti-rabbit IgG (Biocell, Cardiff, UK; 1:50). As negative controls, preimmune serum and PBS were applied instead of the primary antibody.

	Results

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Purification of 29-kD Protein (CAH II)
To minimize contamination with serum components, only dropped-out rat coagulating gland secretion was used for purification. It contains five major proteins of 115 kD, 80 kD, 70 kD, 65 kD (transglutaminase; Seitz et al. 1991 ), and 29 kD. Transglutaminase and 115-kD protein could be separated by fractionated ammonium sulfate precipitation (30–51%). The supernatant contained 29-kD, 70-kD, and 80-kD proteins, which were further separated by size-exclusion chromatography on Superdex 200 columns. The resulting 29-kD protein-enriched fractions were finally purified to homogeneity using anion exchange chromatography. SDS- PAGE and Coomassie staining demonstrated a single band of 29-kD protein co-migrating with bovine erythrocyte CAH II as standard (Figure 1). In total, about 500 µg of pure 29-kD protein was isolated from the residual secretion of coagulating glands prepared from 20 adult rats.

View larger version (63K): [in this window] [in a new window]   Figure 1. SDS-PAGE of rat coagulating gland secretion (Lane 1, 15 µg), purified rat CAH II (Lane 2, 2µg), and standard CAH II from bovine origin (Lane 3, 2 µg) stained with Coomassie brilliant blue. Western blot analyses of rat coagulating gland secretion (Lane 4, 15 µg) and bovine CAH II as standard (Lane 5, 2µg) were probed with the antibody against rat CAH II. A pronounced reaction was shown with rat CAH II (29 kD protein), whereas bovine CAH II did not crossreact. Molecular weight markers in kD are indicated at left.

Identification of Rat 29-kD Protein as CAH II
Protein identification was performed by N-terminal amino acid sequence analyses. Sequencing of the undigested protein proved not to be possible, probably because of N-terminal acylation. However, analyses of Lys-C or trypsin peptide fragments (Ile-Thr-Glu-Ala-Leu-His-Ser-Ile-Lys, Glu-Pro-Ile-Thr-Val-Ser-Ser-Glu-Gln-Met-Ser-His-Phe-Arg-Lys, Ala-Val-Gln-His-Pro-Asp- Gly-Leu-Ala-Val-Leu) were completely identical to the amino acids 158–166, 212–226 and 132–142 of rat brain CAH II (Stolle et al. 1991 ).

Isolated secretory 29-kD protein from rat coagulating gland showed enzymatic activity of carbonic anhydrase. In comparison to commercially available CAH II from bovine erythrocytes, the calculated specific activity of 29-kD protein was 50%. Furthermore, high unspecific esterase activity in the same range as control carbonic anhydrases II from bovine and human origin was demonstrated for rat secretory 29-kD protein. This activity assay was utilized to demonstrate enrichment of this enzyme during isolation. We could achieve an enrichment factor of 100 after the final purification step.

The immunological relationship of rat secretory 29-kD protein to CAH isoforms I and II from bovine and human erythrocytes was examined using Western blotting studies. Depending on the degree of sequence homology, our anti-rat CAH II antiserum reacted with similar isoforms of other species. The closely related human CAH II, with a sequence homology of 80%, was strongly positive (Montgomery et al. 1987 ). In contrast, no immmunoreaction was achieved with the human CAH I and bovine CAH II, corresponding to their lower sequence homologies of 57–71% (Figure 1, Table 1) (Sciaky et al. 1976 ; Barlow et al. 1987 ).

Immunohistochemical and Immunoelectron Microscopic Studies
With both immunoperoxidase and immunofluorescence techniques, the apical cell pole and apical protrusions of the epithelial cells were intensively stained with the CAH II antibody. No immunoreactivity was observed in the intraluminal secretion (Figure 2a–c). However, slight nuclear staining was also noted. Control reactions using either preimmune serum or PBS were negative (data not shown).

View larger version (96K): [in this window] [in a new window]   Figure 2. Immunohistochemistry (a–c) on epoxide resin-embedded semithin sections of rat coagulating gland. Strong CAH II immunoreactivity using immunoperoxide (a,b) or fluorescence staining techniques (c) is visible in the aposomes (arrowheads). L, lumen; E, epithelium Bars = 10 µm. Immunoelectron microscopic studies (d–f) using the affinity-purified antibody against CAH II show gold labeling in the cytoplasm (d,e) and the aposomes (f) of rat coagulating gland. No staining is visible in the ER cisternae (d) and in the Golgi apparatus (e). L, lumen; ERC, ER cisternae; R, ribosomes; N, nucleus; MI, mitochondrion; GO, Golgi apparatus;, V, secretory vesicle; AP, aposomes; ST, stalk; arrows (d) mark gold particles. Bar = 1 µm.

Experiments performed at the ultrastructural level using affinity-purified anti-rat CAH II IgG detected strong labeling exclusively in the cytoplasm and inside the apical protrusions of the epithelial cells of coagulating gland. No staining of gold particles was visible within the ER cisternae, all elements of the Golgi apparatus, and in secretory vesicles (Figure 2d–f). Incubating sections with preimmune serum or PBS instead of the primary antibody yielded negative results.

LDH Distribution in Rat Coagulating Gland
Using Western blot analysis, we examined if LDH, a classical cytoplasmic protein, is present in the total secretion of rat coagulating gland. The results clearly localized LDH to the cytosolic fraction of coagulating gland (Figure 3). Only a very faint band of LDH, most likely originating from inevitable cell damage, was visible in the secretion (Figure 3). We therefore concluded that cytoplasmic LDH was not translocated into the blebs for apocrine secretion and that a specific sorting mechanism must exist for cytoplasmic but apocrine-secreted proteins such as CAH II and TGase.

View larger version (40K): [in this window] [in a new window]   Figure 3. Western blot of rat coagulating gland secretion (Lane 1, 15 µg), rat coagulating gland cytoplasm (Lane 2, 15 µg), and LDH from hog muscle (Lane 3, 2 µg) probed with an antibody against LDH.

	Discussion

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In vitro labeling studies by Bartlett et al. 1984 demonstrated two different secretory pathways in isolated rat dorsal prostate cells. DP II, a highly glycosylated protein (115–120 kD), is exported via the slower classical merocrine pathway that can be inhibited by tunicamycin. In contrast, secretion of DP I (later identified to be a transglutaminase of 65 kD) was increased in the presence of tunicamycin. It was concluded that DP I does not pass through the RER and the Golgi apparatus during intracellular transport, but utilizes an alternative export pathway.

These findings are in accordance with our immunohistochemical studies on rat coagulating gland. Here, we could also demonstrate the existence of two different pathways: merocrine processing and export for 115-kD protein (identical to DP II) and the apocrine secretion mode for secretory transglutaminase (65 kD) (Seitz et al. 1990 ; Steinhoff et al. 1994 ). The apocrine pathway is characterized by protein synthesis on polyribosomes in the cytoplasm and exportation via apical blebs. Aumuller and Adler 1979 showed that these protrusions originate at the apical surface of the cells, in particular from microvilli. Bleb formation was androgen-dependent and was completely prevented by orchiectomy and estrogen application (Steinhoff et al. 1994 ).

In this report we describe the identification of a new protein in rat coagulating gland which is secreted by the apocrine export mechanism. The purified protein of 29 kD was identified as CAH II using amino acid sequence analyses, immunological studies, and activity assays. The amino acid sequences of three isolated internal peptides revealed 100% identity to CAH II from rat brain (Stolle et al. 1991 ). Furthermore, an anti-coagulating gland CAH II antiserum reacted with the highly homologous human CAH II (Montgomery et al. 1987 ), whereas no immunological crossreactivity to the less related human CAH I and bovine CAH II was detectable (Barlow et al. 1987 ; Sciaky et al. 1976 ). Enzyme activity assays showed carbonic anhydrase activity and unspecific esterase activity for 29-kD protein.

CAH II is known as a resident cytoplasmic protein of many epithelial cells (Spicer et al. 1982 ; Kaunisto et al. 1990 ; Harkonen et al. 1991 ). Depending on overall cell function, the cytoplasmic CAH supplies either H⁺ ions in exchange for Na⁺ and K⁺ or HCO₃^- in exchange for Cl^- across the apical or basolateral plasma membrane. The only secreted CAH isoform described thus far has been CAH VI from salivary glands. With immuoelectron microscopy, isoform VI was localized in secretory granules and secretion of serous acinar cells of rat submandibular gland and is secreted by the classical merocrine export mode (Ogawa et al. 1992 ). The present report provides strong evidence that 29-kD protein is another secreted CAH isoform. It confirms observations by Kaunisto et al. 1990 , who postulated an export of CAH II from epithelial cells of human seminal vesicle, ampulla, and ductus deferens via apical protrusions. CAH II is co-localized (immunohistochemical data not shown) in identical aposomes and is finally co-secreted with secretory transglutaminase, an enzyme responsible for the formation of the intravaginal coagulation plug directly after copulation (Seitz et al. 1990 ; Steinhoff et al. 1994 ). This physiological function requires that the blebs must be intact during ejaculation to avoid prematurate coagulation in the urethra. It is hypothesized that, after disintegration of the bleb membrane in the vagina, CAH II is released together with secretory transglutaminase. It remains still unclear how CAH II is involved in the pH titration of the semen after deposition in the vaginal tract.

In this report, we described the isolation of CAH II in large quantities from the secretion of rat coagulating gland. The occurrence of CAH II in secretion proves that this protein represents a secretory rather than a cytoplasmic protein. Our immunoelectron microscopic data clearly show that CAH II is localized in the cytoplasm and in protrusions arising from the apical plasma membrane forming intraluminal blebs. In no case were ER cisternae, the Golgi apparatus, or secretory vesicles immunolabeled. These ultrastructural data provide clear evidence that CAH II does not leave the coagulating gland cells by the classical secretory pathway as described for the salivary isoform VI. The morphological data are supported by the fact that the N-terminus of rat CAH II is blocked, most likely in the same way as another apocrine-secreted protein, the TGase (Ho et al. 1992 ). Furthermore, the primary structure of CAH II showed no signal peptide for ER translocation. Lack of the signal peptide and a blocked N-terminus were found to be characteristic for a number of secreted proteins with unknown secretory mechanism, such as the FGFs, PD-ECGF, interleukin 1, parathymosin, prothymosin, ß4-thymosin, and different transglutaminases such as the blood coagulating factor XIII (Muesch et al. 1990 ). Alternative known pathways, e.g., the ATP-dependent ABC transporters (Higgins 1992 ) or -factor-mediated export systems described for yeast cells could be excluded (Anderegg et al. 1988 ). In some of these cases apocrine secretion, which has previously been described in detail for secretory transglutaminase of rat coagulating gland, can be assumed as a potential export pathway (Seitz et al. 1990 ; Steinhoff et al. 1994 ). In addition, apocrine secretion was postulated for MVDP from mouse vas deferens (Manin et al. 1995 ) and CAH II from seminal vesicle, ampulla, and ductus deferens (Kaunisto et al. 1990 ). With immunoelectron microscopy, none of the apocrine exported proteins could be detected in the cisternae of the ER and Golgi apparatus or inside secretory vesicles. In contrast, the cytoplasm and the apical cell protrusions were exclusively labeled.

Our Western blot and immunoelectron microscopic (data not shown) analysis demonstrated that classical resident cytoplasmic proteins, such as LDH, are not secreted via the apocrine blebs. Therefore, the question arises: Why are only some of more than several thousand cytoplasmic proteins translocated into the blebs? A selection mechanism in these epithelial cells should exist that discriminates between resident cytoplasmic and apocrine-secreted proteins. Selection may occur by still unknown signals on the primary structure of apocrine-secreted proteins or by post-translational modifications. In contrast, cytoplasmic proteins could also possess a signal or modification for retention. Furthermore, signals and substituents should bind to sorting receptors. Cell biological studies are under way to identify the molecular details of apocrine secretion.

	Acknowledgments

Supported by SFB 286 grant B6 and Au 48/12-1 and 2.

We thank Mr Hanno Welker for sequence analyses and Mr Gerhard Jennemann for photographic work.

Received for publication June 24, 1997; accepted November 18, 1997.

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