Original Paper Renal Physiol Biochem 1992;15:33-40
Department of Pediatrics, and Institute of Forensic Medicine, Electron Microscopy and Organic Chemistry, University of Vienna. Austria
Key Words Glomerular basement membrane Collagen type IV ds-Hydroxyproline Proline analogues
Antigenic Changes of the Glomerular Basement Membrane after Incorporation of Hydroxyproline Isomers
Abstract We investigated antigenic changes of murine glomerular base ment membrane (GBM) collagen type IV after oral feedings of 3-cis- or 4-m-hydroxyproline. As shown by thin layer chro matography and gas chromatographic-mass spectrometric studies, 3-cis- and 4-ds-hydroxyproline were incorporated in to the GBM collagen instead of the normal trans isomer and led to a considerable reduction of the collagen antibody reac tivity on indirect immunofluorescence, passive hemagglutina tion and radioimmunoassay when compared to GBM collagen of control animals. We speculate that antigen modification due to the incorporation of stereoisomers could serve as a model for basement membrane immunopathology.
Introduction Alteration of collagen type IV, the main constituent of the glomerular basement mem brane (GBM), is related to many pathological conditions of the kidney. Genetic disorders like Alport’s syndrome, nail patella syndrome and inflammatory processes like Goodpas ture’s syndrome are well known examples.
Accepted: February 1.1991
However, the molecular basis of collagen changes in these diseases remains unclear, and animal models are needed to answer these questions. The in vivo incorporation of pro line analogues into collagen leads to biochem ical, structural, functional and mechanical changes of the involved proteins [1]. As it is not known whether the incorporation of pro line analogues leads to an altered antigenicity,
G ert Lubec UniversitätsKinderklinik Wahringcr Gürtel 18-20 A-1090 Wien (A ustria)
© 1992 S. Karger AG. Basel 1011 -6524/92/ 0151-0033 $2.75/0
Downloaded by: Stockholm University Library 130.237.165.40 - 12/4/2018 9:26:08 PM
B. Lubeca W. Vycudilikb R. Mallingerc J. Hauslerd C. Popowa G. Lubec
Material and Methods Three groups of 10 inbred female white mice, aged 7 weeks, were fed for a period of 4 weeks either with a 0.1% solution of a'.s-3-hydroxvproline in tap water, with a 0.1% solution of c/s-4-hydroxyproline or with tap water (control group). Animalsof all three groups were kept in cages at 23°C environmental temperature un der normal day/night rhythm and had free access to dry mouse cake. After the 4-week feeding period all ani mals were sacrificed by decapitation. The left kidneys of each group were pooled and assessed thereafter, the right kidneys were used for immunofluorescence stud ies. cis-3-D L -h yd roxyprol i n e and traus-3-D L -h yd roxyproline were synthesized according to Häusler [8], d.v-4-hydroxyproline (Sigma H 1756) and Wmv-4-hy-
34
droxyproline (Sigma H 6002) were purchased. We studied the incorporation of hydroxyproline isomers into GBM collagen by thin layer chromatography (TLC) and gas chromatography/mass spectrometry (GC-MS). Preparation o f Samples and TLC GBM collagen was isolated in each animal group according to Dixit [9], One milligram of GBM collagen type IV was hydrolyzed for 12 h at 105 °C using 6 N hydrochloric acid. Thin Layer Chromatography cis-3- and cis-4 Hydroxyproline were determined by TLC according to the method described by Borel et al. [10]: 250 |tl of trichloroacetic acid (1.2 mol/1) were add ed to the hydrolyzed samples, allowed to pass Dowex 50 W and eluted with 2 ml of 4 molar ammonia. Sam ples were evaporated to dryness on a Pierce Reactitherm Heating Module. After redissolving in 200 pi distilled water. 100 pi of this solution w-ere added to 20 pi o-phthalaldehyde (30% triethylamine/70% etha nol. 8 mg/ml. Pierce). This mixture was incubated in the dark at room temperature for 30 min and evaporat ed until dryness under nitrogen atmosphere. The dry dark residues were dissolved in 20 pi of 7-chloro4-nitrobenz-2-oxa-l,3-diazole in ethanol (8 mg/ml, Sig ma C 5261). After incubation at 40°C in a water bath and centrifugation at 2,000g for 2 min, 10 pi of this reaction mixture were applied onto HPTLC plates (LHP Whatman 4806-710). The development solution consisted of acetone:chloroform:ethanol:triethylamine = 20:60:12.5:7.5. The plates were examined after excitation byU V of363 nm. The 4 above-cited isomers were used as standards. Gas Chromatography/Mass Spectrometry N-trifluoracetyl-O-trimethylsilyl-hydroxyprolinetrimethylsilylesters were prepared for GC-MS accord ing to Donike [11]. After hydrolysis of the GBM prep aration (see above), the aqueous hydrochloric acid was evaporated and the residue kept in vacuo over phosphoropentoxide and potassium hydroxide for 24 h to remove traces of water and HC1. The dried residue was mixed with 100 pi of N-methyltrisilyl-trifluoroacetamide (Pierce 48910) and heated to 80°C in a septum closed vial. After I h of occasional shaking, the dark red solution was cooled to room temperature. Fifty micro liters of N-methyl-bis-trifluoroacetamide (Pierce 49700) were added and the mixture was heated to 80°C for 5 min. One microliter of the reaction mixture was used for GC-MS analysis. Pure weight amounts of the
Lubec/Vycudtlik/Mallinger/Häusler/ Popow/Lubec
Antigenic Changes of GBM Induced by Hydroxyproline Isomers
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we decided to study the binding of anticollagcn antibodies to basement membrane colla gen containing m-3-hydroxyproline instead of the normal tram stereoisomer. Provided the incorporation of the m-isomer modifies the antigenic properties of collagens, this model could help to explain pathogenetic mechanisms in human renal immunopathologyStereospecific antigen recognition has pre viously been described for warfarin [2], pro pranolol [3] and methadone [4] isomers. Lalezari and Jiang [5] reported about the immuno logical discrimination of beta-D-glucose isomers [3], Voss and Watt [6] investigated the production of antibodies against haptens of different steric orientation, and Heidelberger et al. [7] immunologically differentiated R and S isomers of 4.6-O-dipyruvyl-D-alphamethyl-galactoside. However, to our knowl edge, the immunological discrimination be tween cis and tram stereoisomers has not been described until now. We therefore studied the reactivity of antibodies against collagen type IV in kidney sections of a mouse animal model with stereoisomeric modifications of collagen due to the incorporation of hydroxyproline isomers.
Direct and Indirect Immunofluorescence Direct and indirect immunofluorescence were per formed according to the principle of Coons and Kaplan (12). Kidney frozen sections (thickness 4 (tm)were pre pared on a Leitzcryocut. The tests were read on a Leitz Orthoplan fluorescence microscope at objective mag nifications of 16x. 40x and lOOx, respectively. The examiner had no information about the identification of the samples in order to prevent subjective readings. Direct Immunofluorescence. For direct immunoflu orescence kidney sections were incubated at room tem perature for 30 min with rabbit-antimouse IgG-FITC (United States Biochemical Corporation. USB. Ohio. USA) and washed after the incubation period with buffered isotonic sodium chloride solution for 30 min. The sections were embedded in glycerol and read. Indirect Immunofluorescence. Antisera for indirect immunofluorescence were raised in rabbits and swine by immunization with mouse collagen type IV. isolated after the principle of Dixit (9). Sera were used after immunization and two boosters with the antigen and complete Freund's adjuvant (Difco). Sera were ab sorbed with glutaraldehyde immobilized serum protein and red blood cells. Sera did not react with other tissue constituents but basement membranes in the immu nofluorescence assays. No reactivity with other colla gen types (I. Ill) was observed. For indirect immu
nofluorescence equal serum protein concentrations (12 mg/200 pi) were applied for each frozen section. Kidney sections were incubated at room temper ature for 30 min with rabbit antitype IV collagen serum (serial dilutions of 1:10 to 1:1.000) and washed for 30 min with isotonic buffered saline solution. Thereaf ter the specimens were incubated for 30 min with goat antirabbi! IgG-FITC (Behring Werke, Marburg, GFR). Washing and embedding was done as cited above. The same procedure was performed using swine antitype IV collagen serum with its corresponding con jugate, rabbit antiswine IgG-FITC (Nordic. USA). Electron Microscopical Examination Biopsies from kidney cortex were fixed immediate ly by immersion in glutaraldehyde (2.5% in 0.2 M phos phate buffer. pH 7.2), for at least 2 h. Thereafter the aldehyde was washed out with phosphate buffer and postfixation was carried out in 1% osmium tetroxide in Michaelis buffer for 2 h [13], The specimens were de hydrated in a series of graded ethanols and finally in propylene oxide. Then they were embedded in Epon812. Sections were cut on a Reichert OMU2 ul tramicrotome. Semithin sections were stained with al kaline toluidine blue O [ 14). Thin sections were stained with uranyl acetate and lead citrate [15], The sections were examined on a Zeiss EM9S2 electron microscope. Passive Hemagglutination Collagen was isolated from pooled kidneys of each group and subjected to collagenolysis in order to break the helical conformation as described [16]. Collagenase digested collagen was adjusted to a protein concentra tion of 1 mg/ml and coupled to red blood cells according to the method of Gold and Fudenberg [17], Geometric dilutions up to 1:1,024 of the sera used in the immu nofluorescence experiments were applied onto micro titer plates and hemolysis was judged by a noninformed investigator. Radioimmunoassay (RIA) Collagen of c/.v-3-hydroxyproline fed animals and of control animals was compared in a commercially avail able RIA for collagen type IV (RIA type IV collagen NCI kit. Behring Werke, Marburg, FRG). One micro gram of collagens isolated from the kidneys was used, serial dilutions of 1:0 to 1:10 were prepared and mea sured. The standard sera delivered with the RIA kit were used for calibration of the assay.
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3- and 4-, cis- and iw/w-hydroxyproline isomers were derivatized in the same way. Appropriate solutions in cyclohexane served as standards for semiquantitative determination in the GBM hydrolysates. Two GC-MS methods were applied to prove the identity of the isom ers: (1) monitoring of the M-15 fragment ion (m/e 356) single ion monitoring (SIM), and (2) taking full mass spectra with a frequency of I s/ntass decade along with the gas chromatographic separation of the derivatives. Gaschromatographic separation was performed on a 25 m fused silica capillary, coated with polymethylsiloxane (HP Ultraphase, 0.31 ID); the oven temper ature was raised from 100 to 200°C at a rate of 10°C/min ; the temperature of the injection port and transfer line was kept at 250°C. The sample was injected splitlessly. The outlet of the separation capillary was connected via a 0.1-mm internal diameter fused silicia capillary to the double focussing mass spectrometer (Varian MAT 112). The resolution was tuned to 1,500, the accuracy of the mass determination in the SIM mode set to 10 ppm. The output of the instrument (70 eV, positive El spec tra) was managed by a L A BC O M 1computer MSS/GB.
SIC F:C0L1
Fig. 1. Results of GC-MS in vestigations: SIM spectrum (m/e 356) of hydrolyzed collagen type IV sample from a control ani mal. Peaks from left to right: 3transA-trans- and 4-e/.s-hydroxyproline (HP), x = Time; y = rela tive intensities.
S IC F :C O L 1 C
Results Incorporation of the hydroxyproline iso mers was demonstrated by TLC. gas chroma tography and mass spectrometry. TLC of the hydrolyzed collagen preparations from mouse
36
kidneys showed the presence of trans-3- and fra/w-4-hydroxyproline in all three groups. cis-3- and cis-4 hydroxyproline were detected only in the collagen hydrolysates of the corre sponding groups. The samples spiked with the corresponding analogues confirmed the chro-
Lubec/Vycudilik/Mallinger/Häusler/ Popow/Lubec
Antigenic Changes of GBM Induced by Hydroxyproline Isomers
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Fig. 2. Results of GC-MS in vestigations: SIM spectrum of a hy drolyzed collagen type IV sample from a 3-d.v-hydroxyproline treat ed animal. Peaks from left to right: 3-trans-, 4-trans-, 3-cis-, and 4-cishydroxyproline. x = Time: y = rel ative intensities.
matographical positions, i.e. the identity of the bands. Separation of the 4 derivatized isomeric hydroxyprolines was obtained due to suffi ciently different gas chromatographical reten tion times (fig. 1). The El-mass spectra of the 3- and 4-hydroxyproline isomers differed in respect to an intense ion fragment at m/e 147 (CH3) 5 Si 20 and at m/e 164, respectively. The difference of the retention times and of the mass spectra allowed a doubtless identifica tion of the isomers and therefore the assign
ment of modified collagens with incorporated cis-3- orm-4-hydroxyproline (fig. 1, 2). SIM of the M15 ion fragment m/e 356 as well as full mass spectra during gas chroma tographic separation demonstrated that nor mal GBM collagen type IV did not contain the cis isomers whereas the GBM of the isomer fed animals contained the corresponding cis isomers. Following the SIM screening for the isomers their identity was also guaranteed by coinjection of pure standard solutions in order to compensate for minor inevitable deviations
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Fig. 3. Indirect immunofluo rescence staining pattern of glo merular loops. Porcine anticolla gen type IV as antibody, rabbit an tiswine IgG-FITC as conjugate. Titer 1:512. Leitz microscope. xlOO.
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of the gas chromatographic retention times. On direct immunofluorescence no immuno globulin deposition was detected in any of the specimens neither along the GBM nor at any other epitope of the kidney. Results of indi rect immunofluorescence tests are given in ta ble 1: rabbit as well as porcine antisera against collagen type IV showed high titers against the GBM of kidneys of mice fed with cis-3-, rà-4-hydroxyproline and control animals (fig. 3). However, kidneys of the cis-3- and rà-4-hydroxyproline fed animals showed a markedly decreased immunoreactivity with both antisera. Examination of renal glomerula by elec tron microscopy showed no differences be tween sections of the cis-3- and c/s-4-hydroxyproline fed and the control animals. How
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1:0 1:1 1:2 1:3 1:4 1:5 1:6 1:7 1:8 1:9 1:10 Collagen solution
ever, we found numerous secretory droplets in the cytoplasmatic processes and in the perinu clear cytoplasm of podocytes of the isomer fed but not in the control animals. On passive hemagglutination, rabbit antibodies reacted with type IV collagen up to a titer of 1:256 in the control animals, up to 1:16 in the m-3hydroxyproline fed animals and up to 1:32 in the m-4-hydroxyproline fed animals. Porcine antibodies reacted with degraded collagen of normal mice up to a titer of 1:512. and with the cis-3- and m-4-hydroxyproline fed animals up to a titer of 1:64. The RIA performed with serial dilutions of collagen solutions showed significantly lower counts for collagen con taining civ-3-hydroxyproline compared with the control animals (fig. 4).
Lubec/Vycudilik/Mallinger/Häusler/ Popow/Lubec
Antigenic Changes of GBM Induced by Hydroxyproline Isomers
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Fig. 4. RIA of collagen type IV of untreated animals (broken line with dots) and of collagen type IV with incorporated 3-ra-hydroxyproline (broken line with circles).
Table!. Results of indirect immunofluorescence investigations (n = 5) Kidneys control animals
cw-3-hydroxyproline animals
c/.v-4-hydroxyproline animals
Porcine anticollagen IV
1:1.000 each
1:32 1:100 1:16 1:100 1:100
1:32 1:100 1:32 1:100 1:64
Rabbit anticollagen IV
1:1.000 each
1:32 1:32 1:32 1:32 1:16
1:32 1:32 1:16 1:32 1:16
Mouse control
negative
negative
negative
Mouse fed m-3-hydroxyproline
negative
negative
negative
Mouse fed d.s-4-hydroxyproline
negative
negative
negative
Discussion Comparing kidney collagen preparations of mice fed with cis-3- and d.y-4-hydroxyproline analogues and of untreated mice, we found clear differences of the immunoreactivity. This was demonstrated on indirect immu nofluorescence, passive hemagglutination, and RI A. These antigenic changes may be ex plained either by a change in the primary structure or by a change in the conformation of the collagen molecules following the incor poration of the cis stereoisomers. A change in the sequential antigenicity is highly probable as the degraded and denaturated collagen molecules showed decreased antibody bind ing in the passive hemagglutination test. Moreover, the decreased antigen reactivity in the RIA of a collagen split product NCI sup ports the hypothesis of sequential antigenic changes. However, this does not exclude that conformational antigenic changes of the
GBM collagen were responsible for the differ ences in the indirect immunofluorescence as say in addition. A change in the antigenicity of the collagen due to the incorporation of proline analogues could have led to the formation of autoanti bodies. This was excluded in our animals by negative results on indirect immunofluores cence. The lack of an autoimmune response to the altered collagen could be explained by ge netic reasons, by induction of tolerance or by the inaccessibility of GBM collagen to the im mune system. However, as we investigated the IgG response only, we cannot exclude an autoimmune reaction involving other immu noglobulin classes. As we found no inflamma tory reaction on morphological examination, a cell-mediated autoimmune mechanism seems highly improbable. Antigenic changes of the GBM due to in vivo modification of collagen have not been shown previously. As we have shown by GC-
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Serum
MS studies that the proline analogues fed to growing mice were in fact incorporated into the GBM collagen, the modification of the collagen molecules must have occurred dur ing the translational process. This implicates the known fact that the transfer RNA for pro lines is not stereospccific [1|. We speculate that our observation of anti genic changes of GBM collagen due to oral
feeding of amino acid analogues could serve as a model for basement membrane pathology and possibly as a model of antigenic modifica tions of other proteins. This could be of bi ological significance as other amino acid ana logues have been found in animal and human nutrition [1. 18].
1 Rosenthal. G. A.: Plant nonprotein amino and imino acids, pp. 232-247 (Academic Press. New York 1982). 2 Cook, E.C.; Ballentine, N.H.; Seiltzmann, T.B. et a!.: Warfarin enantiomer disposition: determina tion by stereoselective radioimmu noassay. J. Pharmacol, exp. Ther. 210:391-399 (1979). 3 Kawashima. K.: Levy, A.: Spcctor. S.: Stereospecific radioimmunoas say for propranolol isomers. J. Pharmacol, exp. Ther. 196:517-523 (1976). 4 Bartos, F.: Olsen. G .D .: Leger, R.N.: Bartos, D.: Stereospecific antibodies to methadone. Res. Commun. Chem. Pathol. Pharma col. 16: 131-142(1977). 5 Lalezari. P.: Jiang. A.F.: Carbohy drate specific antibodies in normal human sera. Vox Sang. 47: 146-152 (1984). 6 Voss, E.W .H.; Watt, R.M.: Steric orientation of hapten groups and the effect of antibody reactivity. Immunochemistrv 14: 237-247 (1977). 7 Heidelberger, M.: Kvarnstroem, 1.: Eriksen, J. et a!.: Immunochemical determination of the configuration of a haptenic substituent. Proc. natl. Acad. Sci. USA 77:4244-4253 (1980).
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8 Häusler. J.: Darstellung von ax und Irans C3 substituierten Prolinver bindungen. Liebigs Ann. Chem. 1073-1092(1981). 9 Dixit. S.N.: Isolation and charac terization of two alpha chain size collageneous polypeptide chans C and D from glomerular basement membranes. FEBS Lett. 106: 379387(1979). 10 Borei. J.P.: Chanard, J.: Randoux. A.: Determination of 3 hvdroxyproline in urine: in Lubec. Methods in nephrology, pp. 22-29 (Libbey, London 1983). 11 Donike. M.: N-Trifluoroacetyl-Trimethylsilyl-Phenolalkylamine. Darstellung und massenspezifischer gaschromatographischer Nachweis. J. Chromatogr. 103: 91-96 (1975). 12 Coons, A.: Kaplan. M.H.: The lo calization of antigens in tissue cells. J. exp. Med. 9 1 :1-16(1950). 13 Palade. G.E.: A study of fixation for electron microscopy. J. exp. Med. 95:285-293 (1952). 14 Trump. B. E: Smuckler. E. A.: Benditt, E.P.: A method for staining epoxy sections for light microscopy. J. Ultrastruct. Res. 5: 343-351 (1961).
15 Reynolds. E. S.: The use of lead ci trate at high pH as an electron opaque stain in electron microsco py. J. Cell Biol. 17: 208-214 (1963). 16 Poteat, II.: Lubec. G.: The action of hydrolytic enzymes on the isolat ed glomerulus: in Lubec, Hudson. Glomerular basement membrane, pp. 41-48 (Libbey. London 1985). 17 Gold, E.R .: Fudenberg. H.H: Chromic chloride: a coupling re agent for passive hemagglutination reactions. J. Immun. 99: 859-869 (1967). 18 Hatanaka. S.I.: Niimura. Y.; Taniguchi. K. et al.: Specific amino acids in some edible mushrooms. Mushroom Sci. (Tokyo) 9: 809-817 (1984).
Lubec/Vycudilik/Mallinger/Häusler/ Popow/Lubec
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References
Department of Pediatrics, and Institute of Forensic Medicine, Electron Microscopy and Organic Chemistry, University of Vienna. Austria
Key Words Glomerular basement membrane Collagen type IV ds-Hydroxyproline Proline analogues
Antigenic Changes of the Glomerular Basement Membrane after Incorporation of Hydroxyproline Isomers
Abstract We investigated antigenic changes of murine glomerular base ment membrane (GBM) collagen type IV after oral feedings of 3-cis- or 4-m-hydroxyproline. As shown by thin layer chro matography and gas chromatographic-mass spectrometric studies, 3-cis- and 4-ds-hydroxyproline were incorporated in to the GBM collagen instead of the normal trans isomer and led to a considerable reduction of the collagen antibody reac tivity on indirect immunofluorescence, passive hemagglutina tion and radioimmunoassay when compared to GBM collagen of control animals. We speculate that antigen modification due to the incorporation of stereoisomers could serve as a model for basement membrane immunopathology.
Introduction Alteration of collagen type IV, the main constituent of the glomerular basement mem brane (GBM), is related to many pathological conditions of the kidney. Genetic disorders like Alport’s syndrome, nail patella syndrome and inflammatory processes like Goodpas ture’s syndrome are well known examples.
Accepted: February 1.1991
However, the molecular basis of collagen changes in these diseases remains unclear, and animal models are needed to answer these questions. The in vivo incorporation of pro line analogues into collagen leads to biochem ical, structural, functional and mechanical changes of the involved proteins [1]. As it is not known whether the incorporation of pro line analogues leads to an altered antigenicity,
G ert Lubec UniversitätsKinderklinik Wahringcr Gürtel 18-20 A-1090 Wien (A ustria)
© 1992 S. Karger AG. Basel 1011 -6524/92/ 0151-0033 $2.75/0
Downloaded by: Stockholm University Library 130.237.165.40 - 12/4/2018 9:26:08 PM
B. Lubeca W. Vycudilikb R. Mallingerc J. Hauslerd C. Popowa G. Lubec
Material and Methods Three groups of 10 inbred female white mice, aged 7 weeks, were fed for a period of 4 weeks either with a 0.1% solution of a'.s-3-hydroxvproline in tap water, with a 0.1% solution of c/s-4-hydroxyproline or with tap water (control group). Animalsof all three groups were kept in cages at 23°C environmental temperature un der normal day/night rhythm and had free access to dry mouse cake. After the 4-week feeding period all ani mals were sacrificed by decapitation. The left kidneys of each group were pooled and assessed thereafter, the right kidneys were used for immunofluorescence stud ies. cis-3-D L -h yd roxyprol i n e and traus-3-D L -h yd roxyproline were synthesized according to Häusler [8], d.v-4-hydroxyproline (Sigma H 1756) and Wmv-4-hy-
34
droxyproline (Sigma H 6002) were purchased. We studied the incorporation of hydroxyproline isomers into GBM collagen by thin layer chromatography (TLC) and gas chromatography/mass spectrometry (GC-MS). Preparation o f Samples and TLC GBM collagen was isolated in each animal group according to Dixit [9], One milligram of GBM collagen type IV was hydrolyzed for 12 h at 105 °C using 6 N hydrochloric acid. Thin Layer Chromatography cis-3- and cis-4 Hydroxyproline were determined by TLC according to the method described by Borel et al. [10]: 250 |tl of trichloroacetic acid (1.2 mol/1) were add ed to the hydrolyzed samples, allowed to pass Dowex 50 W and eluted with 2 ml of 4 molar ammonia. Sam ples were evaporated to dryness on a Pierce Reactitherm Heating Module. After redissolving in 200 pi distilled water. 100 pi of this solution w-ere added to 20 pi o-phthalaldehyde (30% triethylamine/70% etha nol. 8 mg/ml. Pierce). This mixture was incubated in the dark at room temperature for 30 min and evaporat ed until dryness under nitrogen atmosphere. The dry dark residues were dissolved in 20 pi of 7-chloro4-nitrobenz-2-oxa-l,3-diazole in ethanol (8 mg/ml, Sig ma C 5261). After incubation at 40°C in a water bath and centrifugation at 2,000g for 2 min, 10 pi of this reaction mixture were applied onto HPTLC plates (LHP Whatman 4806-710). The development solution consisted of acetone:chloroform:ethanol:triethylamine = 20:60:12.5:7.5. The plates were examined after excitation byU V of363 nm. The 4 above-cited isomers were used as standards. Gas Chromatography/Mass Spectrometry N-trifluoracetyl-O-trimethylsilyl-hydroxyprolinetrimethylsilylesters were prepared for GC-MS accord ing to Donike [11]. After hydrolysis of the GBM prep aration (see above), the aqueous hydrochloric acid was evaporated and the residue kept in vacuo over phosphoropentoxide and potassium hydroxide for 24 h to remove traces of water and HC1. The dried residue was mixed with 100 pi of N-methyltrisilyl-trifluoroacetamide (Pierce 48910) and heated to 80°C in a septum closed vial. After I h of occasional shaking, the dark red solution was cooled to room temperature. Fifty micro liters of N-methyl-bis-trifluoroacetamide (Pierce 49700) were added and the mixture was heated to 80°C for 5 min. One microliter of the reaction mixture was used for GC-MS analysis. Pure weight amounts of the
Lubec/Vycudtlik/Mallinger/Häusler/ Popow/Lubec
Antigenic Changes of GBM Induced by Hydroxyproline Isomers
Downloaded by: Stockholm University Library 130.237.165.40 - 12/4/2018 9:26:08 PM
we decided to study the binding of anticollagcn antibodies to basement membrane colla gen containing m-3-hydroxyproline instead of the normal tram stereoisomer. Provided the incorporation of the m-isomer modifies the antigenic properties of collagens, this model could help to explain pathogenetic mechanisms in human renal immunopathologyStereospecific antigen recognition has pre viously been described for warfarin [2], pro pranolol [3] and methadone [4] isomers. Lalezari and Jiang [5] reported about the immuno logical discrimination of beta-D-glucose isomers [3], Voss and Watt [6] investigated the production of antibodies against haptens of different steric orientation, and Heidelberger et al. [7] immunologically differentiated R and S isomers of 4.6-O-dipyruvyl-D-alphamethyl-galactoside. However, to our knowl edge, the immunological discrimination be tween cis and tram stereoisomers has not been described until now. We therefore studied the reactivity of antibodies against collagen type IV in kidney sections of a mouse animal model with stereoisomeric modifications of collagen due to the incorporation of hydroxyproline isomers.
Direct and Indirect Immunofluorescence Direct and indirect immunofluorescence were per formed according to the principle of Coons and Kaplan (12). Kidney frozen sections (thickness 4 (tm)were pre pared on a Leitzcryocut. The tests were read on a Leitz Orthoplan fluorescence microscope at objective mag nifications of 16x. 40x and lOOx, respectively. The examiner had no information about the identification of the samples in order to prevent subjective readings. Direct Immunofluorescence. For direct immunoflu orescence kidney sections were incubated at room tem perature for 30 min with rabbit-antimouse IgG-FITC (United States Biochemical Corporation. USB. Ohio. USA) and washed after the incubation period with buffered isotonic sodium chloride solution for 30 min. The sections were embedded in glycerol and read. Indirect Immunofluorescence. Antisera for indirect immunofluorescence were raised in rabbits and swine by immunization with mouse collagen type IV. isolated after the principle of Dixit (9). Sera were used after immunization and two boosters with the antigen and complete Freund's adjuvant (Difco). Sera were ab sorbed with glutaraldehyde immobilized serum protein and red blood cells. Sera did not react with other tissue constituents but basement membranes in the immu nofluorescence assays. No reactivity with other colla gen types (I. Ill) was observed. For indirect immu
nofluorescence equal serum protein concentrations (12 mg/200 pi) were applied for each frozen section. Kidney sections were incubated at room temper ature for 30 min with rabbit antitype IV collagen serum (serial dilutions of 1:10 to 1:1.000) and washed for 30 min with isotonic buffered saline solution. Thereaf ter the specimens were incubated for 30 min with goat antirabbi! IgG-FITC (Behring Werke, Marburg, GFR). Washing and embedding was done as cited above. The same procedure was performed using swine antitype IV collagen serum with its corresponding con jugate, rabbit antiswine IgG-FITC (Nordic. USA). Electron Microscopical Examination Biopsies from kidney cortex were fixed immediate ly by immersion in glutaraldehyde (2.5% in 0.2 M phos phate buffer. pH 7.2), for at least 2 h. Thereafter the aldehyde was washed out with phosphate buffer and postfixation was carried out in 1% osmium tetroxide in Michaelis buffer for 2 h [13], The specimens were de hydrated in a series of graded ethanols and finally in propylene oxide. Then they were embedded in Epon812. Sections were cut on a Reichert OMU2 ul tramicrotome. Semithin sections were stained with al kaline toluidine blue O [ 14). Thin sections were stained with uranyl acetate and lead citrate [15], The sections were examined on a Zeiss EM9S2 electron microscope. Passive Hemagglutination Collagen was isolated from pooled kidneys of each group and subjected to collagenolysis in order to break the helical conformation as described [16]. Collagenase digested collagen was adjusted to a protein concentra tion of 1 mg/ml and coupled to red blood cells according to the method of Gold and Fudenberg [17], Geometric dilutions up to 1:1,024 of the sera used in the immu nofluorescence experiments were applied onto micro titer plates and hemolysis was judged by a noninformed investigator. Radioimmunoassay (RIA) Collagen of c/.v-3-hydroxyproline fed animals and of control animals was compared in a commercially avail able RIA for collagen type IV (RIA type IV collagen NCI kit. Behring Werke, Marburg, FRG). One micro gram of collagens isolated from the kidneys was used, serial dilutions of 1:0 to 1:10 were prepared and mea sured. The standard sera delivered with the RIA kit were used for calibration of the assay.
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3- and 4-, cis- and iw/w-hydroxyproline isomers were derivatized in the same way. Appropriate solutions in cyclohexane served as standards for semiquantitative determination in the GBM hydrolysates. Two GC-MS methods were applied to prove the identity of the isom ers: (1) monitoring of the M-15 fragment ion (m/e 356) single ion monitoring (SIM), and (2) taking full mass spectra with a frequency of I s/ntass decade along with the gas chromatographic separation of the derivatives. Gaschromatographic separation was performed on a 25 m fused silica capillary, coated with polymethylsiloxane (HP Ultraphase, 0.31 ID); the oven temper ature was raised from 100 to 200°C at a rate of 10°C/min ; the temperature of the injection port and transfer line was kept at 250°C. The sample was injected splitlessly. The outlet of the separation capillary was connected via a 0.1-mm internal diameter fused silicia capillary to the double focussing mass spectrometer (Varian MAT 112). The resolution was tuned to 1,500, the accuracy of the mass determination in the SIM mode set to 10 ppm. The output of the instrument (70 eV, positive El spec tra) was managed by a L A BC O M 1computer MSS/GB.
SIC F:C0L1
Fig. 1. Results of GC-MS in vestigations: SIM spectrum (m/e 356) of hydrolyzed collagen type IV sample from a control ani mal. Peaks from left to right: 3transA-trans- and 4-e/.s-hydroxyproline (HP), x = Time; y = rela tive intensities.
S IC F :C O L 1 C
Results Incorporation of the hydroxyproline iso mers was demonstrated by TLC. gas chroma tography and mass spectrometry. TLC of the hydrolyzed collagen preparations from mouse
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kidneys showed the presence of trans-3- and fra/w-4-hydroxyproline in all three groups. cis-3- and cis-4 hydroxyproline were detected only in the collagen hydrolysates of the corre sponding groups. The samples spiked with the corresponding analogues confirmed the chro-
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Fig. 2. Results of GC-MS in vestigations: SIM spectrum of a hy drolyzed collagen type IV sample from a 3-d.v-hydroxyproline treat ed animal. Peaks from left to right: 3-trans-, 4-trans-, 3-cis-, and 4-cishydroxyproline. x = Time: y = rel ative intensities.
matographical positions, i.e. the identity of the bands. Separation of the 4 derivatized isomeric hydroxyprolines was obtained due to suffi ciently different gas chromatographical reten tion times (fig. 1). The El-mass spectra of the 3- and 4-hydroxyproline isomers differed in respect to an intense ion fragment at m/e 147 (CH3) 5 Si 20 and at m/e 164, respectively. The difference of the retention times and of the mass spectra allowed a doubtless identifica tion of the isomers and therefore the assign
ment of modified collagens with incorporated cis-3- orm-4-hydroxyproline (fig. 1, 2). SIM of the M15 ion fragment m/e 356 as well as full mass spectra during gas chroma tographic separation demonstrated that nor mal GBM collagen type IV did not contain the cis isomers whereas the GBM of the isomer fed animals contained the corresponding cis isomers. Following the SIM screening for the isomers their identity was also guaranteed by coinjection of pure standard solutions in order to compensate for minor inevitable deviations
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Fig. 3. Indirect immunofluo rescence staining pattern of glo merular loops. Porcine anticolla gen type IV as antibody, rabbit an tiswine IgG-FITC as conjugate. Titer 1:512. Leitz microscope. xlOO.
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of the gas chromatographic retention times. On direct immunofluorescence no immuno globulin deposition was detected in any of the specimens neither along the GBM nor at any other epitope of the kidney. Results of indi rect immunofluorescence tests are given in ta ble 1: rabbit as well as porcine antisera against collagen type IV showed high titers against the GBM of kidneys of mice fed with cis-3-, rà-4-hydroxyproline and control animals (fig. 3). However, kidneys of the cis-3- and rà-4-hydroxyproline fed animals showed a markedly decreased immunoreactivity with both antisera. Examination of renal glomerula by elec tron microscopy showed no differences be tween sections of the cis-3- and c/s-4-hydroxyproline fed and the control animals. How
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1:0 1:1 1:2 1:3 1:4 1:5 1:6 1:7 1:8 1:9 1:10 Collagen solution
ever, we found numerous secretory droplets in the cytoplasmatic processes and in the perinu clear cytoplasm of podocytes of the isomer fed but not in the control animals. On passive hemagglutination, rabbit antibodies reacted with type IV collagen up to a titer of 1:256 in the control animals, up to 1:16 in the m-3hydroxyproline fed animals and up to 1:32 in the m-4-hydroxyproline fed animals. Porcine antibodies reacted with degraded collagen of normal mice up to a titer of 1:512. and with the cis-3- and m-4-hydroxyproline fed animals up to a titer of 1:64. The RIA performed with serial dilutions of collagen solutions showed significantly lower counts for collagen con taining civ-3-hydroxyproline compared with the control animals (fig. 4).
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Fig. 4. RIA of collagen type IV of untreated animals (broken line with dots) and of collagen type IV with incorporated 3-ra-hydroxyproline (broken line with circles).
Table!. Results of indirect immunofluorescence investigations (n = 5) Kidneys control animals
cw-3-hydroxyproline animals
c/.v-4-hydroxyproline animals
Porcine anticollagen IV
1:1.000 each
1:32 1:100 1:16 1:100 1:100
1:32 1:100 1:32 1:100 1:64
Rabbit anticollagen IV
1:1.000 each
1:32 1:32 1:32 1:32 1:16
1:32 1:32 1:16 1:32 1:16
Mouse control
negative
negative
negative
Mouse fed m-3-hydroxyproline
negative
negative
negative
Mouse fed d.s-4-hydroxyproline
negative
negative
negative
Discussion Comparing kidney collagen preparations of mice fed with cis-3- and d.y-4-hydroxyproline analogues and of untreated mice, we found clear differences of the immunoreactivity. This was demonstrated on indirect immu nofluorescence, passive hemagglutination, and RI A. These antigenic changes may be ex plained either by a change in the primary structure or by a change in the conformation of the collagen molecules following the incor poration of the cis stereoisomers. A change in the sequential antigenicity is highly probable as the degraded and denaturated collagen molecules showed decreased antibody bind ing in the passive hemagglutination test. Moreover, the decreased antigen reactivity in the RIA of a collagen split product NCI sup ports the hypothesis of sequential antigenic changes. However, this does not exclude that conformational antigenic changes of the
GBM collagen were responsible for the differ ences in the indirect immunofluorescence as say in addition. A change in the antigenicity of the collagen due to the incorporation of proline analogues could have led to the formation of autoanti bodies. This was excluded in our animals by negative results on indirect immunofluores cence. The lack of an autoimmune response to the altered collagen could be explained by ge netic reasons, by induction of tolerance or by the inaccessibility of GBM collagen to the im mune system. However, as we investigated the IgG response only, we cannot exclude an autoimmune reaction involving other immu noglobulin classes. As we found no inflamma tory reaction on morphological examination, a cell-mediated autoimmune mechanism seems highly improbable. Antigenic changes of the GBM due to in vivo modification of collagen have not been shown previously. As we have shown by GC-
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Serum
MS studies that the proline analogues fed to growing mice were in fact incorporated into the GBM collagen, the modification of the collagen molecules must have occurred dur ing the translational process. This implicates the known fact that the transfer RNA for pro lines is not stereospccific [1|. We speculate that our observation of anti genic changes of GBM collagen due to oral
feeding of amino acid analogues could serve as a model for basement membrane pathology and possibly as a model of antigenic modifica tions of other proteins. This could be of bi ological significance as other amino acid ana logues have been found in animal and human nutrition [1. 18].
1 Rosenthal. G. A.: Plant nonprotein amino and imino acids, pp. 232-247 (Academic Press. New York 1982). 2 Cook, E.C.; Ballentine, N.H.; Seiltzmann, T.B. et a!.: Warfarin enantiomer disposition: determina tion by stereoselective radioimmu noassay. J. Pharmacol, exp. Ther. 210:391-399 (1979). 3 Kawashima. K.: Levy, A.: Spcctor. S.: Stereospecific radioimmunoas say for propranolol isomers. J. Pharmacol, exp. Ther. 196:517-523 (1976). 4 Bartos, F.: Olsen. G .D .: Leger, R.N.: Bartos, D.: Stereospecific antibodies to methadone. Res. Commun. Chem. Pathol. Pharma col. 16: 131-142(1977). 5 Lalezari. P.: Jiang. A.F.: Carbohy drate specific antibodies in normal human sera. Vox Sang. 47: 146-152 (1984). 6 Voss, E.W .H.; Watt, R.M.: Steric orientation of hapten groups and the effect of antibody reactivity. Immunochemistrv 14: 237-247 (1977). 7 Heidelberger, M.: Kvarnstroem, 1.: Eriksen, J. et a!.: Immunochemical determination of the configuration of a haptenic substituent. Proc. natl. Acad. Sci. USA 77:4244-4253 (1980).
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8 Häusler. J.: Darstellung von ax und Irans C3 substituierten Prolinver bindungen. Liebigs Ann. Chem. 1073-1092(1981). 9 Dixit. S.N.: Isolation and charac terization of two alpha chain size collageneous polypeptide chans C and D from glomerular basement membranes. FEBS Lett. 106: 379387(1979). 10 Borei. J.P.: Chanard, J.: Randoux. A.: Determination of 3 hvdroxyproline in urine: in Lubec. Methods in nephrology, pp. 22-29 (Libbey, London 1983). 11 Donike. M.: N-Trifluoroacetyl-Trimethylsilyl-Phenolalkylamine. Darstellung und massenspezifischer gaschromatographischer Nachweis. J. Chromatogr. 103: 91-96 (1975). 12 Coons, A.: Kaplan. M.H.: The lo calization of antigens in tissue cells. J. exp. Med. 9 1 :1-16(1950). 13 Palade. G.E.: A study of fixation for electron microscopy. J. exp. Med. 95:285-293 (1952). 14 Trump. B. E: Smuckler. E. A.: Benditt, E.P.: A method for staining epoxy sections for light microscopy. J. Ultrastruct. Res. 5: 343-351 (1961).
15 Reynolds. E. S.: The use of lead ci trate at high pH as an electron opaque stain in electron microsco py. J. Cell Biol. 17: 208-214 (1963). 16 Poteat, II.: Lubec. G.: The action of hydrolytic enzymes on the isolat ed glomerulus: in Lubec, Hudson. Glomerular basement membrane, pp. 41-48 (Libbey. London 1985). 17 Gold, E.R .: Fudenberg. H.H: Chromic chloride: a coupling re agent for passive hemagglutination reactions. J. Immun. 99: 859-869 (1967). 18 Hatanaka. S.I.: Niimura. Y.; Taniguchi. K. et al.: Specific amino acids in some edible mushrooms. Mushroom Sci. (Tokyo) 9: 809-817 (1984).
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References
