ARTICLE |
Correspondence to: Jürgen Seitz, Dept. of Anatomy and Cell Biology, Robert-Koch-Str. 6, 35037 Marburg, Germany.
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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 46:505511, 1998)
Key Words: secretion, carbonic anhydrase II, carbonate dehydratase II, apocrine, aposome, coagulating gland
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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 acidbase 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
Immunohistochemical studies showed that CAH II isoforms were localized in the gastrointestinal tract, renal tubule, salivary glands, and in the male reproductive tract (
Functionally, the cytoplasmic CAH II isoform acts in concert with integral plasma membrane transport proteins to regulate bicarbonate and proton passage (
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" (
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.
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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 (290310 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 (3051% 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
Amino Acid Sequence Analyses
In situ digestion was performed according to the method of
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 WilburAnderson units. Bovine CAH II was used as a positive control. Unspecific CAH esterase activity was assayed according to
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 (46 week intervals).
Western Blotting
Western blotting was performed as described by
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
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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;
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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 158166, 212226 and 132142 of rat brain CAH II (
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 (
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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 2ac). However, slight nuclear staining was also noted. Control reactions using either preimmune serum or PBS were negative (data not shown).
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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 2df). 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.
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Discussion |
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In vitro labeling studies by
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) (
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 (
CAH II is known as a resident cytoplasmic protein of many epithelial cells (
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 ( (
-factor-mediated export systems described for yeast cells could be excluded (
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.
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Acknowledgments |
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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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