other two being prostatic acid phosphatase and prostate- specific antigen.6'7 The origin in the prostate gland of the f microseminoprotein present in semen has ...
Americanjournal ofPathology, Vol. 137, No. 3, September 1990 Copyright (© American Association ofPathologists
fi Microseminoprotein Is Not a Prostate-specific Protein Its Identification in Mucous Glands and Secretions
Hakan Weiber,* Christer Andersson, Alva Murne,t Gunnar Rannevik, Clas Lindstrom,§ Hans Lilja,t and Per Fernlundt Fronm the D)epartmenits of Surgery, * Clinical Chemistr',t anid Pathology,% u/nihversitw oj'L und, malhn General Hospital, anid the Fertility Research hIstitutej, Malmo, Sivedeni
/ microseminoprotein (3 inhibin, PSP94), an unglycosylated protein of 94 amino acids with unknown function, is one of the predominating proteins in the secretion of the human prostate gland. In this work the authors have demonstrated that the expression of /3 microseminoprotein is not restricted to the prostate and that the protein has a previously unrecognized widespread occurrence in the human body. According to radioimmunoassay, 3 microseminoprotein immunoreactivity ispresent in many nonprostatic bodyfluids. The highest concentrations werefound in secretionsfrom the respiratory tract; in tracheobronchial fluid sometimes even at concentrations comparable to that in seminalplasma (about I g/l). Intermediate concentrations were found in gastricjuice and some samples of secretion from the uterine cervix, whereas tears, saliva, pancreatic juice, bile, and mucus from the colon had low concentrations. According to gel chromatography, the molecular size of the /3 microseminoprotein immunoreactivity present in tracheal fluid, gastric juice, and secretion from the uterine cervix did not differ from that of :3 microseminoprotein in seminalplasma. The /3 microseminoprotein immunoreactive component present in gastric juice had the same amino-terminal amino acid sequence as prostatic / microseminoprotein (14 residues identified in material purified from gastric juice), providingfurther evidencefor chemical identity of a nonprostatic ,3 microseminoprotein with theprostaticprotein. Immunohistochemical staining with affinity-purified antibodies demonstrated the presence of : microseminoprotein in many tissues, including the goblet cells in the tra-
cheobronchial epithelium, tracheobronchial submucosal glands, certain mucosal cells in the antrum of the stomach, some glands ofBrunner in the duodenum, and in parts of the mucosa of the colon. At least in the respiratory tract, the staining was localized to mucus-containing cells. :3 microseminoprotein immunoreactivity also was localized to the cilia of the ciliated epithelium in the respiratory tract, the fallopian tubes, and the Gartner ducts of the uterine cervix. The pattern of tissue distribution of : microseminoprotein found in this work indicates a connection of : microseminoprotein with mucous secretions. (Am J Pathol 1990, 13 7: 593-604) An abundant protein in human seminal plasma is a small unglycosylated protein of 94 amino acids, known as / microseminoprotein.1-5 Its molecular mass is 11 kd, although it appears to be somewhat larger (14-16 kd) on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)2'6'7 and gel chromatography.6'8 The amino acid sequence of / microseminoprotein, which is known from sequence determinations of the protein purified from seminal plasma2-4 as well as of prostate cDNA,5 is unrelated to any other known protein. / microseminoprotein was originally isolated from human semen as a factor inhibiting the pituitary release of follitropin,9 and the protein has also been named / inhibin9 (another name also used is prostatic secretory protein of 94 amino acids.6). The follitropin-releasing inhibitory activity has been questioned, however, because highly purified preparations of the protein were devoid of such activity.10'11 The biologic function of / microseminoprotein is therefore unknown. Supported by grants from the Swedish Medical Research Council (Project B89-13X-7903 and B89-13X-5913), the Faculty of Medicine at the University of Lund, the Cancer Research Fund at Malmo General Hospital, the John and Augusta Persson Foundation, the Anna Lisa and Sven-Eric Lundgren Foundation, the Magnus Bergvall Foundation, the Albert P5hlsson Foundation, and the Greta och Johan Kock Foundation. Accepted for publication April 13, 1990. Address reprint requests to P. Fernlund, Department of Clinical Chemistry, Malmo General Hospital, S-214 01 Malm6, Sweden.
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d microseminoprotein is one of the three most abundant proteins in the secretion of the human prostate, the other two being prostatic acid phosphatase and prostatespecific antigen.6'7 The origin in the prostate gland of the f microseminoprotein present in semen has been proved by several means, including the analysis of prostatic mRNA, the cloning of cDNAs coding for a microseminoprotein from prostatic tissue,512'13 the demonstration that A microseminoprotein is abundant in the prostatic fraction of split ejaculates6'14 as well as in azoospermic ejaculates of subjects with defective seminal vesicles,' and the immunohistochemical demonstration of : microseminoprotein in prostate epithelium.7,15.16 The measurement of prostatic acid phosphatase and prostate-specific antigen in serum has been established in the management of patients with adenocarcinoma of the prostate and it has been suggested to use (3 microseminoprotein as an additional marker for this disease.15-17 Although prostatic acid phosphatase and prostate-specific antigen both seem to be rather specific for prostatic tissue, the observation of almost the same concentration of (3 microseminoprotein in serum from healthy men and women indicates that there are other sources than the prostate gland for this protein.8 Indeed, the presence of : microseminoprotein in the stomach is suggested by previous results of radioimmunoassay of gastric juice18 and by the immunohistochemical demonstration of 3 microseminoprotein in certain epithelial cells of the gastric mucosa.7 The occurrence of (3 microseminoprotein in several other nonprostatic tissues also has been implicated.1920 However, most of the data suggesting a nonprostatic origin of ( microseminoprotein is contradicted by the negative results reported by some investigators.71516 These contradictory reports have prompted us to look for ( microseminoprotein in various body fluids and to reinvestigate the distribution of ( microseminoprotein in nonprostatic tissues.
Materials and Methods Reagents The Vectastain ABC Kit was supplied from Vector Laboratories (Burlingame, CA) and contained blocking normal goat serum, biotinylated anti-rabbit gamma G immunoglobulin (IgG) made in goat, avidin DH, and biotinylated horseradish peroxidase H. Purified (3microseminoprotein and affinity-purified polyclonal rabbit antibodies against ( microseminoprotein were obtained as previously described.7
Body Fluids All fluids were collected in glass tubes without additives. The samples were kept frozen at -20°C until analysis un-
less otherwise stated below. Tears were from five healthy volunteers (two men and three women) after exposure to freshly cut onions. Nasal secretion was collected at two separate occasions from one man with allergic rhinitis. Tracheal secretion was obtained from five subjects undergoing elective abdominal surgery. None had been premedicated with atropine before intubation. The aspiration was performed after intubation, ensuring that only tracheal secretion was to be collected. Saliva was from seven healthy volunteers (three women and four men). Gastric juice was from seven healthy volunteers (three women and four men) who had been fasting for 10 hours before the sampling via a gastric tube was initiated. A pH below 6 and the absence of a greenish color were the criteria used to decide whether the fluid collected was to be regarded as gastric juice. The pH of the gastric juice samples was adjusted to between 8.1 and 8.9 with 1 mol/ (molar) NaOH before analysis. Pancreatic juice was obtained from four patients after partial pancreatic resection for pancreatic cancer. The juice was collected via a catheter left in the pancreatic duct. Bile was aspirated from a gall bladder during an elective cholecystectomy. The resected gall bladder had no macroscopic signs of cholecystitis. Colonic secretion was obtained from three different regions of the colon, the ascending colon during a right-sided hemicolectomy, the sigmoid colon via a terminal sigmoidostomy, and from the rectum via a proctoscope. The secretion was collected by careful scraping of the mucous membrane with a wooden spatula. Perspiration was from six healthy men taking a sauna bath, letting the sweat from one arm drip into a vial. Cerebrospinal fluid samples were from lumbar punction samples sent to the routine laboratory for protein analysis. Only nonpathologic samples (from one man and three women) were chosen for analysis. Amniotic fluid was obtained by amniocentesis from two women both pregnant in the 17th week. Samples of human breast milk were obtained from healthy mothers on a normal diet. Secretion from the uterine cervix was obtained from seven healthy females of fertile age. Human semen was obtained by masturbation from the male in couples undergoing investigation for involuntary infertility. The seminal plasma was collected as the separated supernatant after centrifugation of liquefied semen at 8OOg for 15 minutes. Benzamidine-HCI was added to the seminal plasma to a final concentration of 0.1 mol/l.
Tissues Human specimens for immunohistology were obtained at operation from parotid and accessory salivary glands, esophagus, stomach, duodenum, small and large intestine, rectum, liver, pancreas, gall bladder, kidney, ureter, the tracheal and bronchial part of the upper respiratory tract, lung, skin, skeletal muscle, artery, mammary gland,
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uterus, fallopian tube, ovary, and placenta. Nasal mucous membrane was obtained at postmortem examination about 12 hours after death.
Radioimmunoassay and Estimation of Size of Immunoreactive Components Radioimmunoassay of : microseminoprotein in body fluids and tissue extracts was performed as previously described.8 For three of the body fluids (from trachea, stomach, and uterine cervix), the assay was validated by size estimation of the immunoreactivity by gel chromatography and by checking that dilution curves were parallel to the standard curve. Gel chromatography was performed as described previously8 with dilution of samples in sodium barbital buffer, 75 mmol/l (millimolar), pH 8.6, containing 2.5 g of bovine serum albumin and 0.05 g of sodium azide per liter before application to the column. For other fluids, validation of the assay was restricted to a checking that dilution curves were parallel to the standard curve.
Uppsala, Sweden) as described in Figure 1 A. The part of the effluent from the Superose 12 column containing the peak concentrations of : microseminoprotein according to radioimmunoassay was pooled (Figure 1 A) and applied directly to a ProRPC HR 5/10 reversed phase (C-1 and C8) column (Pharmacia, Uppsala, Sweden), which then was eluted with an acetonitrile gradient as described in Figure 1 B. The part of the effluent corresponding to the single peak of : microseminoprotein according to radioimmunoassay (Figure 1 B) was collected and dried under a stream of N2. One third of the dried material was subjected to reversed phase (C-4) chromatography on a Brownlee Labs BU-300 AquaporeButyl column (Scantec, Partille, Sweden), as described in Figure 1 C. Parts of the effluent from this column corresponding to absorbance peaks in the chromatogram were pooled separately (Figure 1C) and tested for content of ,B microseminoprotein by radioimmunoassay. The only positive pool (D in Figure 1 C) was regarded as pure gastric A microseminoprotein.
Amino Acid Sequencing
Extraction of ,B Microseminoprotein from Gastric Tissue Gastric antral mucosa was obtained at postmortem examination 10 hours after death from a woman (aged 72 years) with no history or signs of gastric disease. The carefully dissected mucosa (5.0 g) was immediately homogenized with a PTA 10-35 Polytron (Kinematica GmbH, Luzern, Switzerland) with cooling in an ice-bath in 40 ml of a 50 mmol/l phosphate buffer, pH 7.5, containing 0.1 mol/l NaCI, 2 mmol/l ethylenediaminetetra-acetic acid (EDTA), 10 mmol/l benzamidine, and 1% (vol/vol) TritonX100 (Sigma Chemical Co., St. Louis, MO). The homogenate was centrifuged for 30 minutes at 30,000g at 4°C. The supernatant was recovered and kept frozen at -20°C until analysis.
Isolation of a Microseminoprotein from Gastric Juice Gastric juice was collected without any additives from a single normal male (aged 37 years) by gastric aspiration after an overnight fast. The aspirate was kept at -200C until use. After thawing, the juice (35 ml) was mixed on an ice-bath with 9 volumes of cold absolute ethanol (about -20°C). After standing at 40C overnight, the precipitated material was collected by centrifugation at 5000g for 20 minutes. The ethanol precipitate was extracted at 0°C with 3 ml of 0.15 mol/l NaCI, and undissolved material was removed by centrifugation. The clear supernatant (3.0 ml) with dissolved material was subjected to preparative gel chromatography on a Superose 12 column (Pharmacia,
The amino-terminal sequence of p microseminoprotein, isolated from gastric juice as described above, was determined in a gas-phase sequencer (Applied Biosystems, Foster City, CA). Phenylthiohydantoin-derivatives were identified in an on-line high-performance liquid chromatograph using a Waters ,uBondapak C-18 reversed phase column and an elution system described previously.
Histologic and Immunohistochemical Techniques Tissue specimens, obtained at operation, were fixed in buffered 10% formalin and subsequently embedded in paraffin. The embedded tissue specimens were selected so that only sections evaluated to be principally microscopically normal were used for immunohistochemical staining. The alcian blue-periodic acid-Schiff (PAS) staining (giving the cells containing acid sialomucin a blue color, those with neutral mucins a red color, and leaving the serous cells virtually unstained) was essentially according to a standard protocol.22 Affinity-purified rabbit antibodies against (3 microseminoprotein were used in an avidin-biotin-complex (ABC) procedure23 to immunostain , microseminoprotein. The working concentration of the antibodies was 0.9 to 1.8 ,ug/ml. The details of the procedure, which employed diaminobenzidine as a chromophore to detect biotinylated horseradish peroxidase, as well as the control experiments to verify the specificity of the immunoreactivity for ( microseminoprotein, have been described previously.7
596 Weiber et al AlP September 1990, Vol. 137, No. 3
0.3
4.0 a
EC:
E
0
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co 0.2
Figure 1. Isolation of 13-microseminoprotei?l immunoreactivity from gastric juice. A: Materialprecipitated with ethanolfrom 3 5 ml ofgastric jui ice u'as dissolved in 3 ml of 0. 15 molll NaCI, anid subjected to preparative gel chromatography at room temperatuire (abouit 20'C) ont a columni (1.6 X 51 cm) of Superose 12, equilibrated anid elluted with a 25 mmol/l TRIS HCI buffer, pH 7.4. The elutioni rate was 0.6 mllminiute. The absorbanice of the effluent was con tinuiiously monitored at 280 iinm (-). Fractionis of 2. 0 ml u'ere collected anid the conicenitrationi of (3 microseminoprotein in each fraction u'as determinied by, radioimmunl1oassay, (- -). The horizonital bar indicatesfractions (fractionZ 34-36)pooled B: The pooled fracforfiurtherpurification. tion1s (volume 6.0 ml)from the gel chromatographj' were applied to a ProRPIC HR 5/ 10 coluimni (0.5 X 10 cm). After ani initial wash uith 90% solvent 1 (0.1% trifluoroacetic acid in water), 10% solvent 2 (0. 1% tri/luoroacetic acid in acetoniitrile) for 40 minlutes, the column uwas eluted uwith a linear gradient from 90% solvent 1-10% solvent 2 to 50% solvent 1-50% solvent 2 durinig a 40-miniu te period. The chromatographj was per/brmeid at room temperature (about 20'C) anid theflowu rate u'as 0. 5 ml/ mi,iu te duirinig the uhole experimenit. The absorbanice of the effluent was conitinuiiouslyS moniitored at 280 nim (-). Durinig applicationt and u'ashing, the effltuent waas collected anidpooled accordinig to the horizontal bars A to C anid thereafter in 15drop fractions (approximately, 0.34 ml). The content of:( microseminoprotei inmimunoreactivity in each fractioni was determinied bj' radioimmunioassay, (- -). The peak fractionis uere pooled as inidicated (bar D) and dried under a streaam of niitrogent. C: Onie third ofthe dried material from the ProRPC chromatography) u'as dissolved in 0.2 ml ofsolvent 1 and applied to a 1BU-30OAquaporeButyl colum1n (2. 1 X 30 mm). After ani inzitial uwash uith 100% sol'vent I for 5.8 minutes, the columni u'as eluted u'ith a linear gradien t from 100% solvent I to 40% solvent 1-60% sol'vent 2 durinig a 60-miniute period. The chroniatographyP wasperformed at a conistanit tenmperatutre of 45'C uith aflou' rate of 0. 1 mll minlute during the whole experiment. The absorbanice of the effluent was monitored at 215 uim (-). Fractionzs of the effluent were collected accordinig to the horizonital barsA to D, and the conicenitrationz ofi3 microseminioproteiii in eachfractioni (0) was determined by radioimmunoassay.
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Results
Radioimmunoassay of f Microseminoprotein in Human Body Fluids and an Extract of Gastric Antral Mucosa The concentration of microseminoprotein was measured by radioimmunoassay in a number of different human body fluids (Table 1). The concentration differed con-
20
40 Elution time (min)
60
0.0
siderably between different fluids; from undetectable, ie, less than 1 gg/l, to more than 1 g/l. Besides seminal plasma the highest concentrations were found in nasal and tracheal secretions. Almost every other fluid examined contained detectable immunoreactive p microseminoprotein, but at a much lower concentration than in the fluids from the respiratory tract. Juice collected from the stomach had an intermediate concentration of ,B microseminoprotein (Table 1).
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Table 1. Cotice,aratioy of 3 micmosemiioproteiin in Hunmatn Bodjy Fluids Determinied by Radioimmunloassay Median concentration Range Body fluid Number of samples (9g/I) (Ag/l) Tears Nasal secretion Tracheal secretion
Saliva Gastric juice Pancreatic juice
39 9900
240,000 13 830
.R':
-eE
Figure 3. Tracheobronichial epithelium oJ ciliated t)pe, uith mutcus-produ(cing goblet cells intterspersed. The cilia and goblet cells show positive immuntostaining (brou'n )Jbr dnmicroseminoprote/in. (X 100) Figure 4. Part of a mixed seromiucouis glantd in the tracheobronichial siub,nucosa. The mutcotus parts shou' positive immunnostaining (brouwn) for ,B microseminioprotein, ubile the serous parts are left unlstainied. (X 100) Figure 5. A ubole mixed seromuzxcouisglanid in the tracheobronichial suibmtucosa. In-
nliolostaininig Jbr d3 microseminloproteini
(vllow-broun) was followed by staining with alciani blutelPAS (bltue). The) mutcouis parts of the gland shou' positive staining
bothb]brfnmicroseminioproteini and mucini. The alciani blue 1'AS staininlg is restricted to the apical part of the cells, possibli' reflectinig a matutrationi process of the muicuts taking place in the cell. The oncocqtelike cells are stained weakly yellow uhile the serotis cells are left unystainied. (X50) even at a concentration comparable to that of seminal plasma. An important question, however, is whether the protein we have demonstrated to be present in various nonprostatic tissues and secretions is identical to the prostate-produced protein. We previously have presented evidence that the : microseminoprotein immunoreactivity measured in human
blood serum, from men and from women alike, represents the same protein that is present in seminal plasma.8 Our evidence included the demonstration that the serum species had the same molecular size on gel chromatography as the prostatic protein and that dilution curves of the serum immunoreactivity were parallel to that obtained with the prostatic protein. In this work, we have obtained the
600 Weiber et al AjP September 1990, Vol 13 7, No. 3
Figure 6. Gastric antral mucosa. Epithelial cells in the region of the mucosa about one third up from the lamina muscularis mucosae shouw positive immunostaining (broun) for f3 microseminoprotein. (X 10) Figure 7. Ciliated epithelium from the fallopian tube. The cilia display positive immunostaining (brown)forf3 microseminoprotein (X 100).
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601 AJP September 1990, Vol. 137, No. 3
same kind of evidence for identity of the protein measured in secretions from bronchi, stomach, and uterine cervix with the ,3 microseminoprotein of seminal plasma, ie, an identical size on gel chromatography and a parallel dilution curve in the immunoassay. Although the validation of the assay for the other body fluids examined in this work was restricted to a check that dilution of the sample gave consistent results, we think it is highly probable that it is the same protein that has been measured in all of the fluids. In addition to having the correct molecular size, we have shown that the component in gastric juice responsible for the f,-microseminoprotein immunoreactivity has an amino-terminal sequence identical to that published for prostatic i# microseminoprotein.24 However, it is possible that some or all of the protein subjected to sequence analysis after its isolation from gastric juice had not been produced locally but represented swallowed saliva or tracheobronchial secretion. No matter what the origin, the finding is a strong proof for a nonprostatic origin in the body of a protein identical to prostatic ,B microseminoprotein. The presence of , microseminoprotein in several different nonprostatic tissues was demonstrated by immunohistochemistry. The immunohistochemical staining used in this work should be highly specific for prostatic ,B microseminoprotein, as the antibodies employed were isolated by affinity chromatography on immobilized ,B microseminoprotein, which was very pure, according to SDS-PAGE.7 The specificity of the staining was confirmed by the total blocking of the staining by purified seminal f microseminoprotein and the lack of staining when nonimmune rabbit serum was used. The presence of p microseminoprotein in nonprostatic tissues has previously been suggested by the detection of p microseminoprotein in gastric juice by radioimmunoassay,18 the histochemical demonstration of f3-microseminoprotein immunoreactivity in gastric mucosa,7 the finding of comparable concentrations of ,B microseminoprotein in serum of men and women,8 and by the demonstration of mRNA for ,B microseminoprotein in the tissues from the stomach and the respiratory tract.13 The testis, the epididymis, the seminal vesicles, the ovary, and the placenta have also been implicated by previous findings to contain ,B microseminoprotein.1920 The failure of other previous immunohistochemical investigations15"16 to detect f microseminoprotein in such nonprostatic tissues, ie, from stomach, pancreas, and colon, that were clearly positive with our procedure, may be explained by differences between the antisera used. ,B microseminoprotein was detected in the majority of the secretions that were examined in this work, although the concentrations varied considerably between different secretions. Only for cerebrospinal fluid and milk did all samples have a concentration of ,3 microseminoprotein below the detection limit of the assay.
Besides seminal plasma, the highest concentrations were found in the secretions of the respiratory tract. This
fact is consistent with the intense immunohistochemical staining observed in the lining epithelium and submucosal glands of the trachea and bronchi. Also, the surface of the lining epithelium was stained, a staining that seemed to be concentrated to the cilia of the epithelial cells. In the lining epithelium and the submucosal glands, : microseminoprotein seemed to be confined to mucus-containing cells. Our failure to detect ,B microseminoprotein by immunohistochemical staining in nasal mucosal tissue, in spite of the fact that nasal secretion had a high concentration of I3 microseminoprotein according to radioimmunoassay, is unexplained. However, the nasal mucosal tissue examined was obtained at a postmortem examination a rather long time after death, and we cannot exclude that postmortem changes have influenced the result. It would be desirable, therefore, to investigate a more native nasal tissue, and also to measure f3 microseminoprotein in nasal fluid from a larger number of individuals. Gastric juice also exhibited a rather high concentration of ,B microseminoprotein. Although we cannot with certainty exclude that all of this 3 microseminoprotein represented contamination from swallowed bronchial secretion, our conclusion that there is a local production of ,B microseminoprotein in the antrum of the stomach is supported by several independent observations. According to radioimmunoassay of a homogenate of gastric antral mucosa, the tissue concentration of ,3 microseminoprotein was about 10 times that in the gastric juice and there were numerous distinct cells in the antral mucosa that stained with the anti-,B-microseminoprotein antibodies. These cells are located close to the tubular crypts leading from the mucosal mucous glands to the gastric lumen. A gastric production of p microseminoprotein is also supported by the demonstration of f microseminoprotein mRNA in gastric antral tissue.'3 The ,B microseminoprotein-containing cells in the antral mucosa had a similar appearance and distribution as the G cells, which are known to produce gastrin, but we have not yet been able to decide whether the two kinds of cells are identical or if the ,B microseminoprotein-containing cells have any other connection to gastrin secretion. The immunohistochemical investigation demonstrated the presence of ,3 microseminoprotein in many different locations in the body, a finding consistent with the detection of f microseminoprotein by radioimmunoassay in the majority of the body fluids investigated in this work. It is also possible to discern a pattern in the distribution of ,B microseminoprotein in the human body. Disregarding some exceptions, it seems as if ,3 microseminoprotein is present either within cells responsible for mucus production, or in cells with an anatomic location close to
602 Weiber et al AlP Septemtber 1990, Vol. 1.3 7, No. 3
the mucus-secreting structures, or on surfaces usually covered by a layer of mucus. This pattern may indicate a connection of 3 microseminoprotein with mucous secretions. Mucus has important functions in the body.25 It acts as a protective layer on many surfaces in contact with the external world and as a lubricant in hollow structures of the body, including the gastrointestinal tract and many secretory ducts. Besides water, mucin is the main component of mucus, but it has not been possible to explain the variation in physicochemical properties of the mucus from different secretions by a variation in the chemical structure of the mucin. It is thus possible that components other than mucin also are important for the properties of mucus. As mentioned, the biologic function of /3 microseminoprotein is as yet unknown, but the association of ,B microseminoprotein with mucous secretions demonstrated in this work may suggest that : microseminoprotein is important for the function of the mucus. For instance, : microseminoprotein may be a factor modifying the properties of the mucus, eg, viscosity or ability to adhere to the surfaces of mucous membranes. It may be noted that in the mucus film covering the ciliated surface epithelium of the trachea and the bronchi, : microseminoprotein is found in that part of the mucus film in which the cilia beat, which is more fluid than the luminal part of the film. Another possible hypothesis is that / microseminoprotein has a protective function for the mucus, eg, by inhibiting endogenous enzymes from leukocytes, which might degrade mucin, or by acting as an antibacterial agent, preventing bacteria from attacking the mucus barrier. In conclusion, we have shown that : microseminoprotein, a predominant protein in the secretions of the prostate gland, also is present in many other tissues and secretions of the human body. It seems to be associated with some mucous secretions, but its biologic function is unknown. The occurrence of /3 microseminoprotein in many tissues other than the prostate gland is a serious objection against the use of the serum concentration of / microseminoprotein as a marker for prostatic disease.
References 1. Tsuda R, Inoue T, Hara M: A seminal plasma specific antigen of prostate gland. Japanese Journal of Legal Medicine 1982,
36:703-709 2. Seidah NG, Arbatti NJ, Rochemont J, Sheth AR, Chretien M: Complete amino acid sequence of human seminal plasma 3-inhibin. FEBS Lett 1984,175:349-355 3. Johansson J, Sheth A, Cederlund E, Jornvall H: Analysis of an inhibin preparation reveals apparent identity between a
4.
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peptide with inhibin-like activity and a sperm-coating antigen. FEBS Lett 1984,176:21-26 Akiyama K, Yoshioka Y, Schmid K, Offner GD, Troxler RF, Tsuda R, Hara M: The amino acid sequence of human 3 microseminoprotein. Biochim Biophys Acta 1985, 829:288294 Mbikay M, Nolet S, Fournier S, Benjannet S, Chapdelaine P, Paradis G, Dub6 JY, Tremblay R, Lazure C, Seidah NG, Chr6tien M: Molecular cloning and sequence of the cDNA for a 94-amino-acid seminal plasma protein secreted by the human prostate. DNA 1987, 6:23-29 Dub6 JY, Frenette G, Paquin R, Chapdelaine P, Tremblay J, Tremblay RR, Lazure C, Seidah N, Chretien M: Isolation from human seminal plasma of an abundant 16 kDa protein originating from the prostate, its identification with a 94-residue peptide originally described as f3-inhibin. J Androl 1987, 8: 182-189 Lilja H, Abrahamsson PA: Three predominant proteins secreted by the human prostate gland. Prostate 1988, 12:2938 Abrahamsson P-A, Andersson C, Bjork T, Fernlund P, Lilja H, Murne A, Weiber H: Radioimmunoassay of # microseminoprotein, a prostatic-secreted protein present in sera of both men and women. Clin Chem 1989, 35:1497-
1503 9. Thakur AN, Vaze AY, Dattatreyamurthy B, Arbatti NH, Sheth AR: Isolation and characterization of inhibin from human seminal plasma. Indian J Exp Biol 1978, 16:854856 10. Kohan S, Froysa B, Cederlund E, Fairwell T, Lerner R, Johansson J, Khan S, Ritzen M, JornvalI H, Cekan S, Diczfalusy E: Peptides of postulated inhibin activity: Lack of in vitro inhibin activity of a 94-residue peptide isolated from human seminal plasma, and of a synthetic replicate of its C-terminal 28-residue segment. FEBS Lett 1986, 199: 242-248 11. Gordon WL, Liu WK, Akiyama K, Tsuda R, Hara M, Schmid K, Ward DN: /3 microseminoprotein (3 mSP) is not an inhibin. Biol Reprod 1987, 36:829-835 12. Brar A, Mbikay M, Sirois F, Fournier S, Seidah NG, Chretien M: Localization of the human prostatic secretory protein PSP94 and its mRNA in the epithelial cells of the prostate. J Androl 1988, 9:253-260 13. Ulvsback M, Lindstrom C, Weiber H, Abrahamsson PA, Lilja H, Lundwall A: Molecular cloning of a small prostate protein, known as / microseminoprotein, PSP94 or ,B-inhibin, and demonstration of transcripts in non-genital tissues. Biochim Biophys Res Commun 1989,164:1310-1315 14. Beksac MS, Khan SA, Eliasson R, Skakkebaek NE, Sheth AR, Diczfalusy E: Evidence for the prostatic origin of immunoreactive inhibin-like material in human seminal plasma. Int J Androl 1984, 7:389-397 15. Doctor VM, Sheth AR, Simha MM, Arbatti NJ, Aaveri JP, Sheth NA: Studies on the immunocytochemical localization of inhibin-like material in the human prostatic tissue: Comparison of its distribution in normal, benign, and malignant prostates. Br J Cancer 1986, 53:547-554 16. Dub6 JY, Pelletier G, Gagnon P, Tremblay RR: Immunohistochemical localization of a prostatic secretory protein of 94 amino acids in normal prostatic tissue, in primary pros-
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17.
18.
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Acknowledgments The authors thank Ingrid Dahlkvist and Elise Nilsson for their expert technical assistance.
