Human Pregnancy Zone Protein and as-Macroglobulin HIGH-AFFINITY BINDING OF COMPLEXES TO THE SAME RECEPTOR ON FIBROBLASTS AND CHARACTERIZATION BY MONOCLONAL ANTIBODIES*

Pregnancy zone protein (PZP) was isolated from late pregnancy serum and examined for binding to normal skin fibroblasts in culture. A high-affinity binding site on these cells is demonstrated for PZP reacted with methylamine. Experiments with a2-macroglobulin (a2M) and PZP, both modified by methylamine, showed this receptor to be identical to the previously characterized receptor for a2M-proteinase complexes Leuven, F., Cassiman, J. J., and den Berghe, H. (1979) J. Biol. Chem. 254, 5155-5160). With available monoclonal antibodies directed toward a2M and prepared toward PZP, only a limited cross-reaction was observed. We obtained a monoclonal antibody which defines a neo-antigenic site on PZP-methyl-amine, completely analogous to the monoclonal anti- body F2B2, which was previously shown to define a neo-antigenic site on a2M complexes (Marynen, P., Van Leuven, F., Cassiman, J. J., and Van den Berghe, H. (1981) J. Immunol. 127, 1782-1786). These results provide evidence for the homologous function of a2M and PZP as proteinase scavengers. The need for an extra proteinase inhibitor of the a2M-type in pregnancy is discussed. The monoclonal antibodies now available will prove helpful in quantitation and eventually isolation of proteinase complexes of a2M and PZP.

These results provide evidence for the homologous function of a2M and PZP as proteinase scavengers. The need for an extra proteinase inhibitor of the a2M-type in pregnancy is discussed. The monoclonal antibodies now available will prove helpful in quantitation and eventually isolation of proteinase complexes of a2M and PZP.
Pregnancy zone protein (PZP),' first described by Smithies (1959), is quantitatively one of the most important pregnancyassociated plasma proteins in humans; at term, plasma levels of 1-2 mg/ml are not uncommon. The characterization of this protein has been hampered by the difficult separation from az-macroglobulin (aZM). Immunoaffinity isolation from latepregnancy serum (Stigbrand et al., 1978;Folkersen et al., 1978) has the drawback that the conditions needed to elute PZP from immobilized antibodies lead to more or less extensive denaturation of the isolated protein. Nevertheless, the main structural characteristics were elucidated denaturation * This work was supported by Grant 3.0055.83 from the National Fund for Scientific Research, Belgium, by Grant "Geconcerteerde Acties" from the Belgian Government, and by a research grant from the American Cystic Fibrosis Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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The abbreviations used are: PZP, pregnancy zone protein; azM, a,-macroglobulin; aZM. MA, a2M reacted with methylamine; PZP . clonal antibodies; SDS-PAGE, sodium dodecyl sulfate-polyacrylam-MA, pregnancy zone protein reacted with methylamine; mAb, monoide gel electrophoresis; HPLC, high pressure liquid chromatography; NHF, normal human fibroblasts. yielded 360-kDa species, which upon reduction dissociated into 180-kDa subunits (Von Schoultz and Stigbrand, 1974;Bohn and Winckler, 1976;Stimson and Farquharson, 1978). These and other characteristics were highly reminiscent of human aZM, the well-known proteinase inhibitor (Van Leuven, 1982;Sottrup-Jensen et al., 1984a). Moreover, determination of part of the primary structure of PZP revealed an extensive sequence homology with a2M (Sottrup-Jensen et al., 1984b). The separation of PZP and azM, the most difficult step in the overall purification, became possible by zincchelate affinity chromatography (Sinosich et al., 1983). While the work described here was in progress, PZP obtained by zinc-chelate affinity chromatography in an active form was established as a proteinase inhibitor, displaying all the salient features of human azM: the clevage of the PZP peptide chain by the proteinase, the residual activity of the proteinase in the complex, and the appearance of free thiol groups (Sand et al., 1985). The latter was confirmed to be derived from an internal thiol ester, located in the same nonapeptide sequence as in azM (Sand et al., 1985;Sottrup-Jensen et al., 1984a).
In this report, we present results which extend the homology of PZP and azM to the physiological level: both proteinase inhibitors, when complexed, bind with similar high affinity to the receptor on human fibroblasts which was previously characterized as specific for a2M complexes (Van Leuven et al., 1978,1979.

DISCUSSION
The results presented here establish PZP as a close functional homologue of azM: both proteins share the same highaffinity receptor expressed on human fibroblasts. This receptor has been characterized as specifically recognizing a2M only after reaction of the latter with a proteinase or with methylamine (Van Leuven et al., 1979, 1982a, 1982bVan Leuven, 1982). Moreover, we have demonstrated that breaking the internal thiol esters in azM (by aminolysis with methylamine) is necessary and sufficient to express the receptor recognition site (Van Leuven et al., 1982a, 1982b. The homology between PZP and azM at the structural level (Sottrup-Jensen et al., 1984b) including the internal thiol esters and binding of proteinases (Sand et al., 1985) is hereby further Portions of this paper (Including "Experimental Procedures," "Results," Figs. 1-8, and Table 1) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-1834, cite the authors, and include a check or money order for $4.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. extended aminolysis of the internal thiol esters in PZP also brings about the expression of a receptor recognition site. Although not explicitly demonstrated here, it is obvious that complexes of proteinases with PZP will be bound to this receptor and cleared by receptor-mediated endocytosis. Preliminary experiments with PZP-trypsin and PZP-chymotrypsin complexes showed little difference from PZP. MA in binding to skin fibroblasts? This would mean that PZP is a proteinase scavenger like azM and uses the same receptormediated pathway for clearance.
The "raison d'6tre" of PZP must then be sought in those characteristics which are different from aZM. The major difference is its temporary appearance in the circulation: in normal conditions, only pregnancy is characterized by highly increased levels of PZP (for review, see Von Schoultz and Stigbrand, 1982). This "acute phase"-like increase in concentration has been interpreted (Sand et al., 1985) to be analogous to acute phase azM homologues in species other than man, e.g. azM in the rat (Gauthier and Mouray, 1976). The similarity is only correct in terms of "induced synthesis" but not where actual triggers are concerned: in rat, the synthesis of acute phase azM is induced by interleukin 1 and macrophagederived factors (Bauer et al., 1984(Bauer et al., , 1985, whereas in humans, the appearance of PZP is triggered by estrogens (Von Schoultz and Stigbrand, 1982, and references therein). A role for PZP in pregnancy would only be meaningful if PZP and a2M are complementary in their ability (in kinetic terms) to inhibit proteinases and mediate their clearance.
Another major difference between azM and PZP is evident from rate electrophoresis: whereas a2M is well-known to be tetrameric (in fact &-dimeric), PZP behaves like a dimer in the native state. Reaction with methylamine but not with trypsin induces further aggregation to species similar in mobility to a2M. It is hard to imagine any physiological consequence, unless major differences in interaction with the receptor between dimeric and tetrameric PZP complexes exist.
No evidence for this was obtained, although this ambiguity in PZP structure poses a problem. Indeed, since the preparations of PZP.MA used in the experiments described contained both dimeric and tetrameric forms, it was impossible to calculate exactly the binding affinity and receptor number. By gel filtration, no separation of the two forms was possible (Sand et al., 1985); which indicates either a highly asymmetric structure for PZP or an association to tetramers in solution. The observation of dimers in rate electrophoresis would then constitute an aberrant behavior of PZP in this system. None of these explanations are satisfactory, however, to account for all observations (Sand et al., 1985). Unless the problem is solved, we cannot decide whether dimer-tetramer formation has any physiological importance.
The cross-reaction with PZP of monoclonal antibodies prepared against aZM was less than expected from the extreme sequence homology. mAb F2B2 and F12A3, which were shown to define epitopes in the receptor recognition site at the carboxyl terminus of azM complexes (Marynen et al., 1981;Van Leuven et al., 1983, 1986b, did not react with PZP or PZP . MA in rate electrophoresis (Fig. 8), radioimmunoassay, or reversed dot blotting (Van Leuven et al., 1986a). Hence, the similar high-affinity receptor binding obviously still allows for highly different immunochemical characteristics of the receptor recognition site in these two homologous proteins.
From a panel of 59 mAb directed toward a2M which we reacted also with azM and/or azM.MA. Two of these were further characterized together with the PZP-MA-specific mAb F57.12F.5. The latter is in fact the equivalent for PZP of mAb F2B2 and F12A3 for azM: they define neo-antigenic sites not present on the native proteins. mAb F57.9Dll and F57.13G5 also define neo-antigenic sites on PZP.MA, but they also react with aZM. MA.
In conclusion, the observations reported here prove that PZP, like a2M, can be modified at the internal thiol esters by methylamine to express a receptor recognition site. The modified PZP shares with a2M. MA the high-affinity receptor on human skin fibroblasts. The homology is also apparent from the reactivity with monoclonal antibodies. Moreover, as with a2M, a mAb defining a neo-antigenic site related to the receptor recognition site on PZP.MA was obtained. These mAb are likely to become useful tools in the quantitation and the isolation of azM-and PZP-proteinase complexes. The demonstration of specific proteinases bound to PZP and to aZM should eventually clarify their physiological role.