Binding of immunoglobulins to the major progesterone-induced proteins secreted by the sheep uterus

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 260, No. 1, January, pp. 208217,1988

Binding of lmmunoglobulins to the Major Progesterone-Induced Proteins Secreted by the Sheep Uterus’

PETER J. HANSEN’ AND GARY R. NEWTON

Department of Dairy Science, University of Florida, Shealy Drive and R&hey Road, Gainesville, Florida 32611

Received May 22, 1987, and in revised form September 4, 1987

We examined the binding of immunoglobulins to the uterine milk proteins, the major progesterone-induced proteins secreted by uterine endometrium of pregnant ewes. Binding was ascertained by measuring binding of ‘251-immunoglobulin to uterine milk proteins that were Western or dot-blotted to nitrocellulose or were coupled to Sepha- rose. The magnitude of binding was greatest for sheep IgM, intermediate for sheep secretory IgA, low for human secretory and serum IgA, and barely detectable for sheep IgG. Binding of IgA and IgM to uterine milk proteins was time and concentration dependent, saturable, inhibited by high ionic strength buffers, and lost due to enzymatic destruction of the Fc portion of the immunoglobulin molecule. In conclusion, the uterine milk proteins preferentially bind IgA and IgM in a species-dependent manner. Such binding may be related to the role of these proteins in the uterus and may make the uterine milk proteins a useful tool for studying or purifying sheep immunoglobu- 1iIlS. 0 1988 Academic Press, Inc.

The uterine milk proteins are a pair of progesterone-induced glycoproteins secreted in copious amounts by uterine endometrium of the pregnant ewe (l-5). They are also present in colostrum (6). These proteins represent an excellent model for studying genie regulation and function of progesterone-induced proteins because of their hormonal control, abun- dance, and ease of purification. Up to gram quantities of UTM-proteins3 can be recovered from the uterus of a single ewe,

i Supported by NIH New Investigator Award HD 20671.

a To whom correspondence should be addressed. 3 Abbreviations used: Ig, immunoglobulin; UTM-

proteins, uterine milk proteins; secIgA, secretory IgA; serIgA, serum IgA; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BSA, bovine serum albumin; PBS-BB, phosphate-buffered saline binding buffer [lo mM potassium phosphate, pH 7.4, containing 0.15 M NaCl, 0.1% (v/v) BSA and 0.5% (v/v) Triton X-100]; ABB, acetate binding buffer [20 mM acetate, pH 5.5, containing 0.1% (v/v) BSA and 0.5% (v/v) Triton X-100].

making these proteins among the most abundant progesterone-induced proteins described. The UTM-proteins exist in two isomeric forms which have molecular weights of 55,000 and 57,000 and similar, highly basic isoelectric points. The isomers are structurally related and probably arise from a single intracellular pre- cursor (5).

A biological role for UTM-proteins has not been described. The proteins have certain features of lysosomal enzymes, in- cluding the presence of mannose 6-phosphate, the so-called lysosomal recognition marker (7), on oligosaccharide chains of UTM-proteins secreted in culture (5). An enzymatic activity has not been associated with the proteins, however. It was once thought that UTM-proteins exerted a sup- pressive effect on activated lymphocytes (8) but it has since been shown that this activity is caused by another basic uterine protein (9). In the present paper, we report that UTM-proteins bind to certain classes of immunoglobulin, in particular IgA and

0003-9861/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

208

IMMUNOGLOBULIN BINDING TO SHEEP UTERINE MILK PROTEINS 209

IgM. Given the importance of IgA in local immunity in mucosal tissues (10) and colostrum (ll), this binding may be biologi- cally important in both uterine fluid and colostrum. The selectivity of immunoglobulin binding displayed by UTM-proteins may make these proteins valuable reagents for studying or purifying sheep immunoglobulins.

EXPERIMENTAL PROCEDURES

Materials. Human secIgA from colostrum and human serIgA were obtained from Calbiochem. Sheep IgM and IgG were from Pel-Freez or Calbio- them. Carrier-free Nal%I was obtained from Amer- sham and Iodo-Gen was from Pierce. Electrophoresis reagents were as described elsewhere (9) and nitrocellulose was purchased from Schleicher & Schuell. Sigma provided CM-cellulose. Other chromatogra- phy products were from Pharmacia. Pierce was used as a source of immobilized papain, and pepsin and pepstatin A were obtained from Sigma. Antisera were products of Pel-Freez. All other reagents were of the highest quality available.

Puri$cation of UTM-proteins. Uterine milk proteins were purified from uterine fluid of pregnant ewes by a combination of ion-exchange chromatogra- phy using CM-cellulose and gel filtration with Se- phacryl S-200 (1.4). Final preparations were greater than 95% pure when analysed by one-dimensional SDS-PAGE, with the major contaminant being lower molecular weight material associated with breakdown products of UTM-proteins (4).

Collection of uterine jlushings from a pseudopregnant gilt. One female pig was injected with estradiol valerate at Days 11-15 of the estrous cycle to prevent regression of the corpus luteum and induce pseudo- pregnancy (12). At Day 90 postestrus, the gilt was laparotomized and the uterus was flushed (13) to ob- tain uterine proteins.

Pur$cation of secIgA from colostrum. Ovine colostrum was collected by manual milking of the udder within 24 h after lambing. Secretory IgA was purified from the fat-free whey fraction by a combination of ammonium sulfate precipitation, chromatog- raphy with Sephacryl S-200, passage through Pro- tein A-Sepharose (to remove IgG), and gel filtration using Sepharose CL-GB. Based on SDS-PAGE, the final preparation ran as secretory IgA and appeared pure. Ouchterlony double diffusion analysis (14) re- vealed a single band when purified IgA was reacted against rabbit antisera to bovine IgA or whole sheep serum.

Protein determination. Protein concentrations were determined using the procedure of Lowry et al. (15) with BSA as the standard.

Iodinations. Protein (20 fig) was reacted for 15 min with 0.5 or 1 mCi ‘=I in tubes coated with 10 pg 1,3,4,6-tetrachloro-3a,6a-diphenylglycouril (Iodo- Gen) in 1 ml of 20 mM potassium phosphate buffer, pH 7.0, containing 0.4 M NaCl. Bound iodine was separated from free by passage through a Pharmacia PD-10 desalting column with 10 mM Tris-HCl, pH 7.5, 0.15 M NaCI, O;l% (w/v) gelatin as the eluant. Specific activities of radioiodinated Ig varied from 19 to 42 &i/fig.

Western blotting of UTM-proteins. Purified UTM- proteins were boiled in 6.25 mM Tris-HCl, pH 6.8, containing 2.5% (w/v) SDS, 5% (w/v) 2-mercap- toethanol, and 15% sucrose and 200-300 fig was streaked across the top of a 14-cm-wide SDS polyacrylamide gel (7.5 or 12.5%, w/v). After electrophoresis (16) and Western blotting (17) onto 0.45 pm nitrocellulose membranes, the resultant blot was cut into strips and blocked with 0.1 to 1.0% (w/v) BSA. Individual strips were incubated with 1251-Ig diluted in incubation buffer (either PBS-BB or ABB) for 4.5 or 24 h. Strips were then washed in incubation buffer for 3-4 h; the wash solution was changed one time. Dried strips were placed with Kodak XAR X-ray film in the presence of enhancing screens to localize binding by autoradiography.

Dot blotting of UTM-proteins. Binding of Ig to UTM-proteins was quantified by measuring binding of UTM-proteins in reactions carried out in a 96-well dot-blotting apparatus (Bio-Dot Apparatus, Bio- Rad). All determinations were done in triplicate. One hundred microliters of UTM-proteins (6.25 to 300 rg/ml) or lysozyme (to monitor nonspecific binding) was applied to a nitrocellulose membrane using the blotting apparatus according to the manufacturer’s instructions. While still in the apparatus, the nitrocellulose was blocked with PBS-3% BSA (w/v) and then incubated with ‘2sI-Ig (100 wl/well) diluted in incubation buffer (generally ABB). After incubation for 2 h at room temperature, the ‘=I-Ig solution was vacuumed through the nitrocellulose and the membrane was washed twice by adding 0.4 ml incubation buffer to each well while the apparatus was under vacuum. Individual dot blots were excised and counted for radioactivity using a y scintillation counter. Specific binding to UTM-proteins was determined by subtracting binding to lysozyme.

Time dependence of binding. The dot blot assay described above was not suitable for long-term incubations because the nitrocellulose membrane would dry out. Therefore, the assay was modified for studies to ascertain the time course of Ig binding. Purified UTM-proteins or lysozyme were dot-blotted as before. The apparatus was then disassembled and the nitrocellulose membrane was blocked in PBS-3% BSA. Individual dot blots of UTM-proteins or lysozyme were excised and placed in 12 X 75-mm tubes containing ‘%I-Ig in a total volume of 0.35 ml ABB.

210 HANSEN AND NEWTON

After incubation for up to 31 h at room temperature, fluid was removed by aspiration and blots were washed three times with 1 ml ABB and counted for radioactivity.

Binding to UTM-proteins conjugated to Sepharose. Purified UTM-proteins were coupled to Sepharose 4B or Sepharose CL-4B via the cyanogen bromide method (18). The final amount of UTM-protein coupled was about 2 mg/ml gel. To a 2-ml column of this material was applied radiolabeled Ig (250,000- 500,000 cpm) diluted in 10 mM potassium phosphate, pH 7.4, containing 0.15 M NaCl and 1% (v/v) Triton X-100. Bound radioactivity was removed using sequential elutions with 0.1 M acetic acid, 50% (v/v) ethylene glycol, and, if necessary, 6 M guanidine-HCl.

Enzyme digestions. For papain digestion of iz51- sheep secIgA, 0.10 ml immobilized papain from Pierce was washed twice and resuspended in a mixture of 0.8 ml of 20 mM potassium phosphate, pH 6.2, 0.4% (w/v) cysteine, and 0.7% (w/v) EDTA and 0.2 ml containing 9 FCi ‘%I-IgA in 10 mM Tris, pH 7.5, 0.15 M NaC1, 0.1% (w/v) gelatin. The reaction proceeded overnight at 37°C and was terminated by centrifuging the mixture and transferring the su- pernatant to a 94 X 1.5-cm column of Sephacryl S-200. Reaction products were eluted from the column using 10 mM Tris, pH 8.2, 0.33 M NaCl.

Pepsin digestions of ‘a51-sheep secIgA and iz51- sheep IgM were done by reacting 1 fig pepsin and 3.6 (IgM) or 9 FCi (IgA) ‘?-Ig in a total volume of 1 ml containing 0.8 ml 20 mM acetate, pH 4.1, and 0.2 ml 10 mM Tris, pH 7.5, 0.15 M NaCI, 0.1% (w/v) gelatin. The reaction proceeded for 5 h at 37°C and was terminated by adding 0.5 ml of 0.10 M Tris-HCI, pH 8.2, and 10 J of a 0.70 mg/ml solution of pepstatin A. Reac- tion products were separated on a 97 X 2.5-cm column of Sepharose CL-6B with 10 mM Tris-HCl, pH 8.2, 0.33 M NaCl as eluant.

Peaks of reaction products resolved by gel filtration were concentrated if necessary [by dialysis against Hz0 (M, cutoff 6000-8000) and lyophiliza- tion], diluted in ABB and tested for ability to bind 30 ng UTM-proteins that were dot-blotted to nitrocellulose.

RESULTS

Binding of immunoglobulins to Western- blotted UTM-proteins. All radioiodinated Igs tested, except for sheep IgG, bound to UTM-proteins that were transferred to nitrocellulose by electroblotting (Fig. 1). For certain blots, binding of Ig to both isomers of UTM-proteins could be resolved (Fig. 1A).

To verify that 1251-Ig did not bind to all proteins transferred to nitrocellulose, ‘%I- human secIgA was incubated with West-

human sheep sheep

set ser set IgA IgA IgA IgM

set W W

A B C

FIG. 1. Autoradiographs of Western blots of UTM- proteins incubated with 1251-Ig. Amount of UTM- proteins transferred to each strip of nitrocellulose after electrophoretic separation on 7.5% (A, B) or 12.5% (C) polyacrylamide gels was 14 (A), 21 (B), or 9 pg (C). Blots were blocked with BSA and then incubated for 4.5 h (A) or overnight (B and C) with 7 X lo6 (A), 1 X lo6 (B), or 0.5 X lo6 cpm/ml (C) of ‘%I-Ig. Incubation buffer was PBS-BB (A) or ABB (B and C). Exposure of X-ray film was for 24 (A, C) or 1 h (B). Note that all ‘%I-Igs except sheep IgG (C) bound to UTM-proteins and that binding of Ig to both isomeric forms of UTM-proteins could sometimes be resolved (arrows in A).

ern blots of unfractionated uterine fluid from a pregnant ewe, uterine flushings from a pseudopregnant gilt, and serum from a nonpregnant ewe (Fig. 2). As determined by autoradiography of the blots, the major proteins reacting with ‘251-IgA were UTM-proteins.

Quantitation of binding using dot-blotted UTM-proteins. This was carried out by incubating 1251-Ig with UTM-proteins transferred to nitrocellulose using a dot-blotting apparatus. Incubations were done di- rectly in the apparatus and nonspecific binding was determined by replacing UTM-proteins with lysozyme, another basic protein. As seen in Fig. 3, binding was concentration dependent with respect to both Ig and UTM-proteins. The binding was also saturable with maximum binding occurring at about 10 pg UTM-proteins (Fig. 3C). Binding was greater for sheep secIgA than for human IgAs (Fig. 3B), and greater for sheep IgM than for sheep sec- IgA (Fig. 3A). Binding of ‘251-sheep IgG


FIG. 2. Autoradiogram of a Western blot of purified UTM-proteins (UTMP), uterine fluid from a pregnant ewe (sheep Ut Fl), uterine flushings from a pseudopregnant gilt (pig Ut Fl), and serum from a nonpregnant sheep (sheep serum) after incubation with iz51-human secIgA. Proteins (25-35 pg/lane) were separated by SDS-PAGE using 10% (w/v) polyacrylamide gels, transferred to nitrocellulose and, after blocking with PBS-0.1% BSA, incubated with ‘%I-IgA (1.6 X lo6 cpm/ml in PBS-0.1% BSA) for 3 h at room temperature. The blot was washed in PBS-BB and exposed to X-ray film for 24 h. Note that the major proteins binding ‘%I-IgA were UTM-proteins (arrow). The lower molecular weight proteins binding IgA in the lane of purified UTM-proteins represent breakdown products of UTM-proteins.

was negligible, being only slightly greater than nonspecific binding (Fig. 3A).

Binding to UTM-proteins conjugated to Sepharose. Radioiodinated human secIgA was found to bind to a column of UTM- proteins conjugated to Sepharose 4B. When 0.5 X lo6 cpm was placed on a 2-ml column, 61% of the radioactivity bound to the column. Using sequential elution, 18% of the bound radioactivity was removed with 0.1 M acetic acid, 1.5% by 50% (v/v) ethylene glycol, and 41% by 6 M guanidine-HCl. The remaining radioactivity was not recovered. In contrast, 85% of ‘251-sheep IgG placed over a column of UTM-proteins conjugated to Sepharose 4B failed to bind and the remaining 15% was not recovered after sequential elution with acetic acid and ethylene glycol.

Time dependence of binding. This was examined for radioiodinated sheep secIgA and IgM. The amount of 1251-Ig bound to dot-blotted UTM-proteins increased with time. For IgM, equilibrium was reached at about 22-28 h (Fig. 4).

Dependence of binding on pH and ionic strength. The effect of pH on binding of sheep secIgA to dot-blotted UTM-proteins was examined using a range of pH’s from 4 to 8. Binding was independent of pH for ‘251-IgA diluted in 10 mM Tris-maleate, pH 5.5-8.0, acetate, pH 4.0-5.5, and phosphate, pH 6.0-8.0 (results not shown). The percentage of radioactivity specifically bound to UTM-proteins was similar regardless of buffer chosen.

The effect of ionic strength on binding of Igs to dot-blotted UTM-proteins is presented in Fig. 5. Binding of both Igs tested (sheep secIgA, sheep IgM) decreased as the concentration of NaCl in incubation buffer increased.

Binding of papain and pepsin-digested IgA. When radioiodinated sheep secIgA that had been incubated at 37°C for 24 h was resolved by gel filtration using Se- phacryl S-200 (Fig. 6A), three peaks of radioactivity could be detected; a peak at the void volume (Fraction l), a peak eluting at a iV& range of 150,000 and 69,000 (Fraction 2), and a peak eluting at the salt volume. These peaks most likely represented di- merit secretory IgA (l), monomeric IgA or degraded IgA fragments (2), and free 1251 or radioiodinated peptide fragments (salt peak).

Incubation of ‘251-IgA with papain or pepsin altered this elution pattern. After papain digestion, the major peak of radioactivity (Fraction 1) eluted at M, 45,000 (Fig. 6B), a molecular weight consistent with this peak representing Fab fragments of IgA. Other peaks resolved were of low molecular weight and presumably represent extensively degraded peptide fragments of IgA. Products of pepsin digestion of IgA were separated by Sepha- rose CL-6B (Fig. 6C). Major peaks resolved included a peak eluting at a molecular weight characteristic of F(ab)2 fragments of IgA (Fraction 2, M, 96,000) and a series of peaks near the salt volume.

HANSEN AND NEWTON

Radloactlvity Added Radioactivity Added

(cpm x 10’7 kpm X l@)

0 . . 0 II 10 1. u

Protein Blotted (pg)

FIG. 3. Concentration dependence of binding of ‘%I-Ig to UTM-proteins. Purified UTM-proteins were dot-blotted to nitrocellulose and incubated for 2 h at room temperature with ‘?-Ig. After they were washed, individual dot blots were excised and counted for radioactivity. Incubation buffer was PBS-BB (A and C) or ABB (B). Data presented in A involved Ig’s of sheep origin incubated at various concentrations with 20 pg UTM-proteins. Data in B were from sheep and human IgAs reacted with 30 lg UTM-proteins. For both A and B, results are expressed as specific binding, calculated by subtracting radioactivity bound to lysozyme from radioactivity bound to UTM-proteins. Average binding to lysozyme (percentage of total radiolabel added) was 4.3 (sheep IgA), 4.1 (sheep IgM), 3.0 (sheep IgG), 3.7 (human secIgA), and 1.9% (human serIgA). Data in C represent binding data for incubations of lzI-sheep IgM (75,000 cpm/well) with varying concentrations of UTM-proteins or lysozyme. Similar results were obtained for sheep IgA (not shown). Binding is plotted as total radioactivity bound to UTM-proteins and lysozyme.

The binding of each of these fractions to UTM-proteins was tested (inset graphs in Fig. 6). The binding of the original preparation of lz51-sheep secIgA was 20.5%, and binding of Fraction 1 of undigested IgA separated by gel filtration was 16.3% (Fig. 6A). No more than 6.1% of the papain or pepsin-digested fragments bound UTM- proteins (Figs. 6B and 6C).

Pepsin digestion of IgM. Chromatogra- phy of undigested ‘251-sheep IgM by Seph-

arose CL-6B (Fig. 7A) resolved two major peaks of radioactivity, one at the void volume (presumably aggregated IgM) and one peak eluting at a volume characteristic of polymeric IgM (just before the 666,000 M, thyroglobulin standard). Pep- sin-digested 1251-sheep IgM was resolved into three fractions (Fig. 7B); Fraction 1 eluted as IgM; Fraction 2 had a broad peak within the molecular-weight range of 42,000-91,000; and Fraction 3 was com-

IMMUNOGLOBULIN BINDING TO SHEEP UTERINE MILK PROTEINS

Time (hr)

FIG. 4. Time dependence of the binding of radioiodinated sheep secIgA and sheep IgM to UTM-proteins. Individual dot blots of UTM-proteins (30 pg) were incubated in 12 X 75-mm tubes with 90,000 (IgA) or 100,000 cpm (IgM) in 0.35 ml ABB for various times at room temperature. Specific binding (percentage of total cpm added) is shown and was calculated by subtracting counts bound to lysozyme. Binding of IgM and IgA was time dependent, with equilibrium for IgM being reached after -22-28 h.

posed of low molecular weight material (kfr < 26,000). When tested for binding to dot-blotted UTM-proteins, both fractions resolved from lz51-sheep IgM bound UTM- proteins (Fig. 7A). The same was true for Fraction 1 resolved from digested 1251- sheep IgM but Fractions 2 and 3 only bound minimally (Fig. 7B).

A

0.1 0.1 0.4

Salt Concentration (Ml Salt Concentration (Ml

213

DISCUSSION

The results presented here indicate that UTM-proteins selectively bind certain classes of immunoglobulins, in particular, sheep IgA and sheep IgM. Binding to UTM-proteins was demonstrated in test systems utilizing UTM-proteins absorbed to nitrocellulose and covalently coupled to Sepharose. Binding of immunoglobulin to UTM-proteins was not simply a nonspecific interaction between iodinated immunoglobulin and other proteins since (i) only IgA and IgM and not IgG bound to UTM-proteins, (ii) binding to lysozyme, another basic protein, was low, and (iii) binding of lz51-human secIgA to proteins in sheep serum or uterine flushings from a pseudopregnant pig was not detected.

As mentioned, the degree of binding varied considerably among immunoglobulins. Binding was greater for IgA and IgM than for IgG and was greater for sheep IgA than for human IgAs. That the binding of UTM-proteins is species specific is further indicated by findings that mono- clonal mouse IgAs were unable to bind col- umns of UTM-proteins coupled to Sepha- rose (P. A. Small and P. J. Hansen, unpublished observations).

In certain cases (i.e., sheep IgM vs sheep secIgA), differences in binding were rela-

6

0 0.1 0.1 Salt Concentration (Ml

FIG. 5. Inhibition of binding of immunoglobulin to UTM-proteins by NaCl. Radiolabeled sheep Igs diluted in ABB containing various concentrations of NaCl were incubated with dot-blotted UTM- proteins (30 pg) for 2 h in a Bio-Dot apparatus. Results shown are specific binding of IgM (A) and IgA (B) to UTM-proteins after subtracting radioactivity bound to lysozyme and expressing results as a percentage of binding in the absence of NaCl.


A B

Elution Volume (ml) Elutlon Volume (ml)

0 too 440

Elutlon Volume (ml)

FIG. 6. Binding of reaction products of enzyme-digested rz?-sheep secIgA to UTM-proteins. For papain digestion, 9 pCi 1251-IgA was incubated overnight at 37°C with 0.1 ml immobilized papain (Pierce) in a total volume of 1 ml containing 0.8 ml of 20 mM phosphate, pH 6.2,0.4% (w/v) cysteine, and 0.7% (w/v) EDTA and 0.2 ml of 10 mM Tris, pH 7.5, 0.15 M NaCl, 0.1% (w/v) gelatin. Pepsin digestion of ‘?-IgA (9 &i) was performed for 5 h at 3’7°C in a 1 ml volume containing 1 pg pepsin, 0.8 ml of 20 mM acetate, pH 4.1, and 0.2 ml 10 mM Tris, pH 7.5,0.15 M NaCl, and 0.1% (w/v) gelatin. In each panel, the main graph displays the elution profile of undigested IgA incubated under the conditions for papain digestion (A), IgA digested with papain (B), or IgA digested with pepsin (C), using a 94 X 1.5-cm Sephacryl S-200 (A and B) or 97 X 2.5-cm Sepharose CL-6B column (C). Elution volumes of molecular weight standards (thyroglobulin, Tg, 660,000; ferritin, Fer, 450,000; alcohol dehydrogenase, AD, 150,000; BSA, 67,000, and chymotrypsinogen A, chymo, 26,000) are indicated by arrows and pooled fractions by horizontal arrows. The inset graphs represent the binding of each fraction diluted in ABB to dot-blotted UTM-proteins (30 pg) after 2 h incubation at room temperature in a Bio-Dot apparatus. Binding data are expressed as percentage of total radioactivity bound to UTM-proteins after subtracting radioactivity bound to lysozyme.

tively slight and could have been caused by differences in iodination damage of the immunoglobulin molecules. Such damage likely occurred because low molecular weight, iodinated fragments could be identified in a preparation of lz51-sheep secIgA separated by gel filtration. Also, binding of immunoglobulin at saturating concentrations of dot-blotted UTM-pro-

teins and to a column of UTM-proteins coupled to Sepharose was less than 100%. Reduced binding of proteins caused by ra- dioiodination damage has also been docu- mented in other systems, such as for binding of luteinizing hormone to membrane receptors (19).

Enzyme digestion studies provided indi- rect evidence that UTM-proteins bind to


1 A E

1.1 - A * ?.I “0

2.2 - o-

$ -pr ““i”‘i”

‘0 1.0 -

~ T- ,.a- C x ,A- 5 - 1.1. i : l.Z- e 6 1.0.

c ; :::

2 a.- 0.z 0.0 ,

0 200 400 MO x-3 400

Elution Volume (ml) Elution Volume (ml)

FIG. 7. Binding of reaction products of pepsin-digested ‘=I-sheep IgM to UTM-proteins. Digestion was carried out by incubating 0.2 ml of issI-IgM (3.6 &i) in 10 mM Tris, pH 7.5, 0.15 M NaCl, 0.1% (w/v) gelatin with 0.8 ml of pepsin (1 fig) in 20 mM acetate, pH 4.1, for 5 h at 37°C. In each panel, the main graph represents the elution profile of undigested IgM incubated under the conditions for digestion (A) or IgM digested with pepsin (B). The column used for separation was Sepharose CL-6B (97 X 2.5 cm). Elution volumes for molecular weight standards (thyroglobulin, Tg, 660,000; ferritin, Fer, 450,000; BSA 67,000; and chymotrypsinogen A, chymo, 26,000) are indicated by arrows and pooled fractions by horizontal arrows. The inset graphs represent the binding of each fraction diluted in ABB to dot-blotted UTM-proteins (30 pg) after 2 h incubation at room temperature in a Bio-Dot apparatus. Binding data are expressed as percentage of total radioactivity bound to UTM- proteins after subtracting radioactivity bound to lysozyme.

the Fc portion of IgA and IgM. Papain and pepsin have been reported to destroy the Fc portion of IgA of other species (20-22), leaving either Fab or F(ab)z fragments, re- spectively. Similar results were obtained in the current experiments for both IgA and IgM as determined by analysis of enzyme digests using gel filtration. None of the fractions characteristic of Fab or F(ab)z fragments or Fc digestion products bound UTM-proteins to the same degree as undigested immunoglobulin. Since both IgA and IgM have J chain associated with the Fc portion of the molecule (23) and pepsin has been reported to destroy J chain on IgM (22), it is possible that UTM-proteins are binding to this moiety. However, binding was also observed to human serIgA, a monomeric IgA with low J chain content (24).

Interactions of UTM-proteins with IgA and IgM are probably ionic in nature because binding was reduced in the presence of high salt concentrations. Furthermore, guanidine-HCl could dissociate 1251- human secIgA bound to a column of

UTM-protein coupled to Sepharose, whereas ethylene glycol had little effect. Preliminary results not presented indicate that iodoacetamide does not reduce binding of 1251-human secIgA to UTM-proteins, suggesting that binding does not involve free sulfhydryl groups.

Binding of immunoglobulins to other proteins in biological fluids is not unique to the UTM-proteins. Besides secretory component, a membrane protein that covalently binds IgA during transport of IgA across epithelia (lo), other IgA binding proteins include albumin (25, 26), lactoferrin (27), peroxidase (28), and a-l an- titrypsin (26). Lactoferrin also binds IgG, albumin, casein, and lactalbumin (29, 30) and peroxidase can associate with IgM, lactalbumin and ovalbumin (25, 28). Many of these protein-protein interactions involve intermolecular disulfide bond for- mation (25-27), though some of the IgA- lactoferrin binding is probably ionic in nature (27). The UTM-proteins are probably distinct from the just-mentioned immunoglobulin binding proteins because of


differences in molecular weight, isoelectric point, degree of glycosylation and amino acid composition (5). In addition, UTM-proteins do not contain iron, unlike lactoferrin, and do not inhibit trypsin (unpublished observations). The binding of UTM-proteins to immunoglobulins also appears to be more specific than for some of these other proteins since binding is class restrictive (IgA and IgM) and species dependent (sheep IgA > human and mouse IgAs).

The physiological significance of immunoglobulin binding to UTM-proteins in the uterine lumen is unknown. The binding affinity may be low in utero because of the effects of salt concentration on binding. Nonetheless, the extremely high concentrations (lop4 M) of UTM-proteins in uterine fluid (4) enhance the probability that most IgA and IgM in uterine secretions are associated with UTM-proteins. Both lactoferrin and peroxidase are more po- tent bactericidal agents when mixed with IgA (28, 31) and it is possible that com- plexes of IgA and UTM-proteins are bactericidal. UTM-proteins possess certain features of lysosomal enzymes (5) and an enzymatic function of UTM-proteins might result in bacterial killing. However, we have been unable to identify an inhibi- tory effect of UTM-proteins on growth of Streptococcus agalactiae or Escherichia co& (unpublished observations). Alter- nately, UTM-proteins might act to prevent effector actions of IgA antibody directed against the fetal placenta which, being al- logeneic, can induce an immune response in the pregnant mother (32). In any case, the immunoglobulin-binding properties of UTM-proteins may make these abundant glycoproteins of practical significance as reagents for purification or analysis of sheep immunoglobulins.

ACKNOWLEDGMENTS

The authors thank P. A. Prudencio and M. V. Leslie for their excellent assistance, M. E. Hissem for pre- paring the manuscript, F. W. Bazer for donation of uterine fluid, and R. M. Roberts, in whose laboratory some preliminary studies were conducted. This is Journal Series No. 8491 of the Florida Agriculture Expt. Sta.

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