Proc. Nati. Acad. Sci. USA
`Vol. 88, pp. 1556-1559, February 1991
`Biochemistry
`
`Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro
`dipeptides and use of these inhibitors to examine the role of DP-IV
`in T-cell function
`GEORGE R. FLENTKE*, EDUARDO MUNOZt, BRIGITTE T. HUBERt, ANDREW G. PLAUTt,
`CHARLES A. KETTNER§, AND WILLIAM W. BACHOVCHIN*¶
`Departments of *Biochemistry and tPathology, Tufts University School of Medicine, Boston, MA 02111; tDepartment of Medicine, Division of
`Gastroenterology, New England Medical Center Hospital, Boston, MA 02111; and §Central Research and Development Department,
`E. I. DuPont de Nemours Company, Experimental Station, Wilmington, DE 19898
`
`Communicated by John D. Roberts, November 26, 1990
`
`ABSTRACT
`Dipeptidyl peptidase IV (DP-IV; dipeptidyl-
`peptide hydrolase, EC 3.4.14.5) is a serine protease with a
`specificity for cleaving Xaa-Pro dipeptides from polypeptides
`and proteins. It is found in a variety of mammalian cells and
`tissues, including those of Iymphoid origin where it is found
`specifically on the surface of CD4' T cells. Although the
`functional significance of this enzyme has not been established,
`a role in T-cell activation and immune regulation has been
`proposed. Here we report that Ala-boroPro and Pro-boroPro,
`where boroPro is the a-amino boronic acid analog of proline,
`are potent and specific inhibitors of DP-IV, having K1 values in
`the nanomolar range. Blocking the N terminus of Ala-boroPro
`abolishes the affinity of this inhibitor for DP-IV, while removal
`of the N-terminal residue, to give boroPro, reduces the affinty
`for DP-IV by 5 orders of magnitude. The dipeptide boronic
`acids exhibit slow-binding kinetics, while boroPro does not. We
`also report here that low concentrations of Pro-boroPro inhibit
`antigen-induced proliferation and interleukin 2 production in
`murine T-cell lines but do not inhibit the response of these T
`cells to the mitogen concanavalin A. These results indicate that
`DP-IV plays a role in antigen-induced, but not mitogen-
`induced, activation of T lymphocytes.
`
`Dipeptidyl peptidase IV (DP-IV; dipeptidyl-peptide hydro-
`lase, EC 3.4.14.5) is a postproline cleaving enzyme with a
`specificity for removing Xaa-Pro dipeptides from the N
`terminus of polypeptides and proteins. DP-IV will also re-
`move Xaa-Ala dipeptides from N termini, but in general this
`reaction is 100- to 1000-fold less efficient (1).
`DP-IV is found in a variety of mammalian cells and tissues.
`It is most abundant in the proximal tubules of the kidneys (2,
`3), intestinal epithelium (4, 5), and placenta (6). It is also
`found in the capillary endothelium (2), blood plasma (2), and
`on the cell surface of certain subsets of T lymphocytes,
`particularly the CD4' helper cells, although a subpopulation
`of human CD8' T cells also exhibits DP-IV activity (7-10).
`Scholz et al.
`(8) have reported that cell-surface DP-IV
`activity among lymphoid cells is associated with the ability of
`a cell to produce interleukin 2 (IL-2).
`A biological function for this enzyme in mammalian sys-
`tems has not yet been established. In yeast, insects, and
`frogs, DP-IV has been shown to be involved in the proteolytic
`processing of a number of bioactive peptides and proteins.
`These include, for example, the proteolytic processing of (i)
`an extracellular protease from Yarrowia lipolytica (11), (it)
`the potent neurotoxin melittin from bee venom (12), (iii) a
`family of antibacterial peptides from insects referred to as
`cecropins (13), and (iv) peptides of unknown function found
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertisement"
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`in frog skin that exhibit homologies with mammalian hor-
`mones and neurotransmitters (14).
`In mammalian systems, a number of different biological
`functions for DP-IV have been proposed, ranging from di-
`gestion (15) and amino acid salvage (16), because of its
`occurrence in the intestine and kidney, to an involvement,
`through proteolytic processing, in fibronectin-mediated cell
`movement and adhesion (17). Because of the high frequency
`of Gly-Pro sequences in collagen, Hopsu-Havu et al. (18)
`suggested that this enzyme may play a role in collagen
`metabolism or catabolism. DP-IV in human plasma has been
`shown to catalyze the removal of an N-terminal Tyr-Ala
`dipeptide from growth hormone-releasing factor (19), and this
`cleavage results in the inactivation of this hormone (20).
`The most intriguing of the biological functions thus far
`suggested for DP-IV comes from Schon et al. (21), who have
`proposed that DP-IV is involved in T-cell activation and
`regulation of T-cell proliferation. This proposal is based on
`experiments in which the addition of DP-IV inhibitors and
`DP-IV antibodies to cell culture systems suppressed mitogen-
`and alloantigen-induced T-cell proliferation, IL-2 production
`(22), and impaired T-cell-directed B-cell differentiation and
`immunoglobulin production (23, 24). Circumstantial support
`for this proposal comes from the observation that a number
`of cytokines have DP-IV susceptible Ala-Pro N-terminal
`sequences, as this suggests a plausible mechanism for the
`proposed involvement of DP-IV in T-cell activation events.
`These cytokines include IL-1f3, IL-2, granulocyte-macroph-
`age colony-stimulating factor, erythropoietin, and macroph-
`age inflammatory protein la, recently reported to be identical
`to stem cell inhibitor. None of these cytokines, however, has
`yet been demonstrated to be a substrate for DP-IV or to have
`altered biological properties with the N-terminal Ala-Pro
`dipeptide deleted.
`Inhibitors for DP-IV, more potent and specific than those
`now available, should prove useful in establishing the bio-
`logical role or roles of this enzyme. Prospects for obtaining
`such inhibitors seemed particularly promising on the basis of
`our work with another class of post-proline-cleaving serine
`proteases, the IgA proteases from Neiserria gonorrhoeae and
`Hemaphilus influenzae. Peptides containing the a-amino
`boronic acid analog of proline (boroPro) as the C-terminal
`amino acid inhibit these enzymes with K, values in the
`nanomolar range (25). Here we report that dipeptides con-
`taining boroPro as the C-terminal residue are indeed potent
`inhibitors of DP-IV, having Ki values as low as 2 X 10-9 M
`and that these inhibitors are effective in inhibiting antigen-
`induced, but not mitogen-induced, lymphocyte proliferation
`
`Abbreviations: DP-IV, dipeptidyl peptidase IV; IL-2, interleukin 2;
`boroPro, a-amino boronic acid analog of proline; Boc, butoxycar-
`bonyl.
`ITo whom reprint requests should be addressed.
`
`1556
`
`AstraZeneca Exhibit 2008
`Mylan v. AstraZeneca
`IPR2015-01340
`
`Page 1 of 4
`
`

`
`Biochemistry: Flentke et al.
`and IL-2 production in cultures of murine CD4' T-helper
`type 1 cells and hybridomas.
`
`METHODS
`Materials. DP-IV was purified from porcine kidneys as
`described (3). The enzyme activity was determined using
`Ala-Pro-p-nitroanilide as substrate, as detailed in the legend
`of Fig. 1. BoroPro, Ala-boroPro, Boc-Ala-boroPro (Boc,
`butoxycarbonyl), and Pro-boroPro were synthesized essen-
`tially as reported (25).
`K1 Values. The rates of DP-IV-catalyzed hydrolysis of
`Ala-Pro-p-nitroanilide were determined at three to five con-
`centrations of each of the boronic acid inhibitors. The reac-
`tion rates observed for Ala-boroPro (Fig. 1B) and for Pro-
`boroPro (data not shown) are nonlinear, while those observed
`for boroPro are linear (Fig. lA). After 10 min, the nonlinear
`reaction rates (Fig. 1B) become linear. The linear portion of
`the rate curves can be duplicated by incubating the enzyme
`with Ala-boroPro or Pro-boroPro for 10 min before adding
`substrate. The K, values reported in Table 1 for Ala-boroPro
`and Pro-boroPro were obtained from the linear rates ob-
`served after incubation of enzyme with inhibitor and thus
`represent final K1 values. We have not yet analyzed the
`nonlinear portion of the rates to obtain initial K, values.
`BoroPro exhibits linear inhibition kinetics and was treated as
`a standard rapid equilibrium competitive inhibitor in arriving
`at the K, value reported in Table 1.
`The K, values for Ala-boroPro and Pro-boroPro likely
`underestimate the true affinity of these inhibitors for DP-IV.
`Both inhibitors are unstable in aqueous solutions of neutral
`pH values. Ala-boroPro decomposes with a half-life between
`2 and 30 min depending on pH. Pro-boroPro is substantially
`more stable with a half-life ofat least 1.5 hr. The slow-binding
`inhibition kinetics of these inhibitors together with their
`instability in aqueous solution would combine to yield the
`linear rates prior to the establishment of true equilibrium
`between free and enzyme-bound inhibitor. K, values obtained
`under these conditions must underestimate the true Ki.
`Cell Lines. The murine CD4' Th-1 clone D1.1 (specific for
`rabbit IgG in the context of I-Ad) and the CD4+ T-cell
`hybridoma 2B4 (specific for cytochrome c in the context of
`I-Ak) were obtained from A. Abbas (Harvard Medical
`School) and A. Korman (Whitehead Institute), respectively.
`D1.1 cells were stimulated every 2 weeks with antigen and
`
`A
`
`0.6
`
`0.5
`
`0.4
`
`t 0.3
`CN
`
`o0.0mM
`
`0.01mM
`
`/0/0/2m
`
`0To
`v
`
`Proc. Natl. Acad. Sci. USA 88 (1991)
`
`1557
`
`Table 1.
`
`Inhibition constants of boronic acid inhibitors of DP-IV
`Ki, nM
`Inhibitor
`>1,000,000*
`N-Boc-Ala-boroPro
`BoroPro
`110,000
`Ala-boroPro
`2
`Pro-boroPro
`3
`*No inhibition detected.
`irradiated I-Ad+ splenocytes serving as antigen-presenting
`cells. Supernatant from rat spleen cells that had been stim-
`ulated with Con A for 48 hr was used as a source of
`lymphokines. The hybridoma 2B4 was maintained at a con-
`centration of 2 x i0s cells per ml in RPMI 1640 medium
`containing 10o fetal calf serum (HyClone Laboratories).
`Assays of Ceil Proliferation and IL-2 Production. D1.1 cells
`were purified on a Ficoll/Hypaque gradient (Pharmacia) at
`least 2 weeks after the last stimulation with antigen. The cells
`were then cultured in 96-well plates, 5 x 104 cells per well,
`in RPMI 1640 medium supplemented with 2 mM L-glutamine,
`1 mM Hepes, 50 mM 2-mercaptoethanol, antibiotics, and
`various amounts of Pro-boroPro. The cells were incubated
`for 1 hr prior to the addition of5 x i01 irradiated I-Ad+ spleen
`cells plus either 200 gg of antigen per ml (rabbit IgG) or 5 Ixg
`of Con A per ml. Twenty microliters offetal calf serum (1%o)
`was added to each well. The cultures were incubated for 60
`hr at 370C, and [3H]thymidine (0.5 ,Ci per well; 1 Ci
`37
`GBq) was added for the final 12 hr of culture. Radioactivity
`incorporated into DNA was measured by liquid scintillation
`counting.
`The 2B4 hybridoma was stimulated as described above for
`the D1.1 cell line, except that irradiated I-Ak+ spleen cells
`were used as the antigen-presenting cells and cytochrome c
`was used as antigen. The cells were incubated for 24 hr prior
`to assaying for IL-2 production. IL-2 production was deter-
`mined in a bioassay using the HT-2 indicator cell line, which
`proliferates in response to IL-2. Aliquots of supernatants
`from the D1.1 and 2B4 cell cultures were added to cultures of
`HT-2 cells (104 cells per well) and incubated for 24 hr with
`[3H]thymidine (0.5
`GCi per well) added for the final 6 hr.
`
`RESULTS AND DISCUSSION
`Inhibition of DP-IV. Ala-boroPro, the peptide boronic acid
`corresponding to the classic substrate for DP-IV, is a potent
`0.61 B
`
`0
`
`2
`
`4
`
`8
`
`10
`
`12
`
`6
`6
`Time, min
`Time, min
`Progress curves for DP-IV inhibitors. Rapid equilibrium vs. slow-binding inhibitors. The reaction solution consisted of 50 .mol of
`FIG. 1.
`sodium Hepes (pH 7.8), 10 jumol of Ala-Pro4-nitroanilide, 6 milliunits of DP-IV, and 2% (vol/vol) dimethylformamide in a total vol of 1.0 ml.
`The reaction was initiated by addition of the enzyme. Rates were measured at 250C. The concentrations of inhibitors are shown on the graphs.
`(A) BoroPro. (B) Ala-boroPro.
`
`0
`
`2
`
`4
`
`12
`
`Page 2 of 4
`
`Page 2 of 4
`
`

`
`Proc. Natl. Acad. Sci. USA 88 (1991)
`
`-A
`
`Am
`
`6
`
`16-6 61-516-4
`16-7
`Pro-boroPro, M
`
`50 -
`
`40-
`
`° 30-
`
`20-
`
`10
`
`xE
`
`IL-2 secretion by a murine CD4+ T-cell hybridoma 2B4
`FIG. 3.
`in response to specific antigen or mitogen. Cells (5 x 104 per well)
`were cultured in the presence of5 x 105 irradiated (2000 R) syngeneic
`antigen-presenting cells plus either specific antigen (A) (cytochrome
`c; 5 AM) or mitogen (o) (Con A; 2 jg/ml) for 16 hr. The supernatant
`was then removed and tested for IL-2 content in a bioassay on HT-2
`indicator cells (104 cells per well). The inhibitor Pro-boroPro was
`added at the indicated concentrations 1 hr before addition of antigen
`or mitogen. HT-2 cell background was 3087 ± 987 cpm.
`
`These results were somewhat unexpected in light of the
`previous reports by Schon and coworkers (21-23) that DP-IV
`inhibitors suppress both antigen- and mitogen-induced T-cell
`proliferation and IL-2 production. The reasons for this dis-
`crepancy are not clear at present. However, Pro-boroPro is
`4-5 orders of magnitude more potent, and probably corre-
`spondingly more specific, an inhibitor of DP-IV than those
`used previously. We therefore believe the results reported
`here to be a more reliable indicator of the role of DP-IV in
`T-cell function. Nevertheless, the present results lend sup-
`port to the overall hypothesis proposed by Schon and co-
`workers (21-23) that DP-IV is involved in T-cell activation,
`even though they conflict with respect to the details of the
`role that DP-IV plays.
`The concentration of Pro-boroPro needed to effect 50%
`inhibition of proliferation and of IL-2 production, -5 x 10-7
`M, is -100-fold greater than the Ki for inhibition of DP-IV by
`this inhibitor. This should, however, not be considered a
`discrepancy. The experiments illustrated in Fig. 2 required
`3-day incubations. As described earlier, Pro-boroPro has a
`half-life of 1.5 hr and in these experiments only a single dose
`of inhibitor was given at the beginning of the incubation.
`Thus, the difference between K1 for enzyme inhibition and
`inhibition of proliferation may reflect the instability of the
`inhibitor. Alternatively, the total amount of DP-IV present
`may significantly exceed the K1 value of 10-9 M. Still another
`possible explanation is that nearly complete inhibition of
`DP-IV activity is necessary before suppression of T-cell
`activation is observed, and such complete inhibition ofDP-IV
`would require 1O-7 M inhibitor even if the total DP-IV
`concentration is small enough to ignore. It is not possible that
`early inhibition of DP-IV is sufficient to suppress antigen-
`induced proliferation, whereas longer term inhibition is nec-
`essary to suppress mitogen-induced proliferation because
`maintaining the inhibitor concentration at high levels by daily
`additions of inhibitor does not affect mitogen-induced pro-
`liferation (results not shown). Nor is it likely that the com-
`bination of antigen-stimulation plus treatment with inhibitor
`induces toxicity, whereas treatment with Con A and inhibitor
`does not because Pro-boroPro suppression of antigen-
`stimulated T cells can be reversed by treatment with IL-2
`(results not shown). Thus, the combination of antigen and
`inhibitor is not toxic in these cells and there is no reason to
`believe that it would be to T-cell clones. Although it is well
`established that T-cell mitogens act through the T-cell recep-
`tor to induce proliferative signals, it is also likely that they
`
`1558
`
`Biochemistry: Flentke et al.
`inhibitor of DP-IV, having a Ki value of 2 x 10-9 M (Table 1).
`Blocking the N terminus of this inhibitor (e.g., N-Boc-Ala-
`boroPro; Table 1) abolishes the affinity, demonstrating that a
`free, positively charged amino group is essential for enzyme
`recognition and binding. The Ki of 3 x 10-9 M for Pro-
`boroPro demonstrates that DP-IV tolerates an imino group in
`place of the amino functional group on the N terminus as well
`as the substitution of a proline side chain in place of the
`alanine methyl group. This shows that the S2 specificity
`subsite is not highly restrictive, a result that correlates with
`the known substrate specificity for Xaa-Pro dipeptides (1,
`26). Although DP-IV will accept nearly any amino acid at the
`N terminus, interactions between this amino acid and the
`enzyme are critical for binding. This is illustrated by the
`105-106 decrease in affinity on going from Ala-boroPro or
`Pro-boroPro to boroPro itself (Table 1).
`Fig. 1 shows that Ala-boroPro and Pro-boroPro, but not
`boroPro, exhibit slow-binding kinetics. This correlates with
`previous studies on the inhibition ofother serine proteases by
`peptide boronic acid inhibitors, which have shown that
`boronic acid analogs of substrates tend to exhibit slow-
`binding kinetics. Boronic acid analogs of nonsubstrates or
`poor substrates may still be inhibitors, but they usually
`exhibit normal inhibition kinetics (27).
`Although the inhibition kinetics reported here were carried
`out on DP-IV isolated from pig kidneys, we have confirmed
`that Pro-boroPro and Ala-boroPro inhibit DP-IV from human
`placenta equally well.
`Effect of Pro-boroPro on T-cell Activation in Murine Lym-
`phocyte Cell Culture Systems. Figs. 2 and 3 summarize our
`findings on the effects ofthe dipeptide boronic acid inhibitors
`on T-cell activation. Fig. 2 shows that Pro-boroPro is a potent
`inhibitor of antigen-induced (rabbit IgG) proliferation of
`murine CD4' T helper cells (D1.1), exhibiting a dose-
`response curve with 50% inhibition occurring at submicro-
`molar concentrations of the inhibitor. In contrast, this com-
`pound is not effective in inhibiting Con A-induced prolifer-
`ation even at much higher concentrations. Fig. 3 shows that
`Pro-boroPro is also more effective in inhibiting antigen-
`induced than mitogen-induced IL-2 production. A similar
`specific inhibition of antigen-induced IL-2 production was
`also observed in D1.1 cell cultures (data not shown).
`
`120 -
`
`100 -
`
`o
`C-ct
`I1)
`.-
`
`80-
`
`60-
`
`e 40-
`
`20 -
`
`I .
`
`0
`
`I
`
`10-7
`10-6
`Pro-boroPro, M
`
`lo-,
`
`1o-4
`
`Proliferative response of a murine CD4+ T-helper type 1
`FIG. 2.
`cell line D1.1. Cells (5 x 104 per well) were cultured in the presence
`of 5 x 101 irradiated (2000 R; 1 R = 0.258 mC/kg) syngeneic
`antigen-presenting cells plus either specific antigen (A) (rabbit IgG;
`200 ,ug/ml) or mitogen (o) (Con A; 2 g/ml) for 3 days. The cells were
`pulsed during the last 6 hr of culture with [3H]thymidine. The
`inhibitor Pro-boroPro was added at the indicated concentrations 1 hr
`before addition of antigen or mitogen. Cells cultured in medium only
`gave 425 ± 4 cpm. The results shown are of a single representative
`experiment, but these results have been reproduced 10 times with an
`SD always '20%o in triplicate assays.
`
`Page 3 of 4
`
`Page 3 of 4
`
`

`
`Biochemistry: Flentke et al.
`
`Proc. Natl. Acad. Sci. USA 88 (1991)
`
`1559
`
`bind to and activate other accessory molecules, such as
`VLA3 and VLA5, which allows them to override the inhib-
`itory effects ofDP-IV inhibition. In contrast, antigen-induced
`proliferation might lack such alternative pathways.
`Because of their high affinity and specificity, the dipeptide
`prolyl boronic acid inhibitors should prove helpful in unrav-
`eling the role played by DP-IV in T-cell activation and in
`defining underlying molecular mechanisms. Should DP-IV be
`confirmed to play the role in antigen-induced T-cell activation
`as the results reported here indicate, these inhibitors may
`also prove useful therapeutically in modifying or controlling
`the immune response.
`
`This work was supported by National Institutes of Health Grants
`GM 27927 (W.W.B.), DE 07257 (A.P.), and Al 23031 (B.T.H.).
`
`1.
`
`2.
`3.
`
`4.
`
`5.
`
`6.
`
`7.
`8.
`
`9.
`
`10.
`
`11.
`
`Heins, J., Welker, P., Schonlein, C., Born, I., Hartrodt, B.,
`Neubert, K., Tsuru, D. & Barth, A. (1988) Biochim. Biophys.
`Acta 954, 161-169.
`Gossrau, R. (1985) Histochem. J. 17, 737-771.
`Wolf, B., Fischer, G. & Barth, A. (1978) Acta Biol. Med. Ger.
`37, 409-420.
`Svensson, B., Danielsen, M., Staun, M., Jeppesen, L., Noren,
`0. & Sjostrom, H. (1978) Eur. J. Biochem. 90, 489-498.
`Corporale, C., Fontanella, A., Petrilli, P., Pucci, P., Molinaro,
`M. F., Picone, D. & Auricchio, S. (1985) FEBS Lett. 184,
`273-277.
`Puschel, G., Mentlein, R. & Heymann, E. (1982) Eur. J.
`Biochem. 126, 359-365.
`Ansorge, S. & Ekkehard, S. (1987) Acta Histochem. 82, 41-46.
`Scholz, W., Mentlein, R., Heymann, E., Feller, A. C., Ulmer,
`A. J. & Flad, H. D. (1985) Cell. Immunol. 93, 199-211.
`Mentlein, R., Heymann, E., Scholz, W., Feller, A. C. & Flad,
`H. D. (1984) Cell. Immunol. 89, 11-19.
`Feller, A. C., Heijnen, C. J., Ballieux, R. E. & Parwaresch,
`M. R. (1982) Br. J. Haematol. 51, 227-234.
`Matoba, S. & Ogrydziak, D. M. (1989) J. Biol. Chem. 264,
`6037-6043.
`
`12.
`
`13.
`
`14.
`
`15.
`
`16.
`
`17.
`
`18.
`
`19.
`
`20.
`
`21.
`
`22.
`
`23.
`
`24.
`
`25.
`
`26.
`
`27.
`
`Kreil, G., Haiml, L. & Suchanek, G. (1980) Eur. J. Biochem.
`111, 49-58.
`Boman, H. G., Boman, I. A., Andreu, D., Li, Z., Merrifield,
`R. B., Schienstedt, G. & Zimmerman, R. (1989) J. Biol. Chem.
`264, 5852-5860.
`Mollay, G., Vilas, U., Hutticher, A. & Kreil, G. (1986) Eur. J.
`Biochem. 160, 31-35.
`Morita, A., Chung, Y.-C., Freeman, H. J., Erikson, R. H.,
`Sleisenger, M. H. & Kim, Y. S. (1983) J. Clin. Invest. 72,
`610-616.
`Miyamoto, Y., Ganapathy, V., Barlas, A., Neuber, K., Barth,
`A. & Leibach, F. H. (1987) Am. J. Physiol. 252, F670-F677.
`Hanski, C., Huhle, T. & Reutter, W. (1985) Biol. Chem.
`Hoppe-Seyler 366, 1169-1176.
`Hopsu-Havu, V. K., Rintola, P. & Glenner, G. G. (1968) Acta
`Chem. Scand. 22, 299-308.
`Frohman, L. A., Downs, T. R., Heimer, E. P. & Felix, A. M.
`(1989) J. Clin. Invest. 83, 1533-1540.
`Frohman, L. A., Downs, T. R., Williams, T. C., Heimer,
`E. P., Pan, Y.-C. & Felix, A. M. (1986) J. Clin. Invest. 78,
`906-913.
`Schon, E., Mansfeld, H. W., Demuth, H. U., Barth, A. &
`Ansorge, S. (1985) Biomed. Biochim. Acta 44, K9-K15.
`Schon, E., Demuth, H. U., Eichmann, E., Horst, H.-J., Kor-
`ner, E.-J., Kopp, J., Mattern, T., Neubert, K., Noll, F., Ulmer,
`A. J., Barth, A. & Ansorge, S. (1989) Scand. J. Immunol. 29,
`127-132.
`Schon, E., Jahn, S., Kiessig, S. T., Demuth, H.-U., Neubert,
`K., Barth, A., Von Baehr, R. & Ansorge, S. (1987) Eur. J.
`Immunol. 17, 1821-1826.
`Gruber, M., Scholz, W. & Flad, H. D. (1988) Cell. Immunol.
`113, 423-434.
`Bachovchin, W. W., Plaut, A. G., Flentke, G. R., Lynch, M.
`& Kettner, C. A. (1990) J. Biol. Chem. 265, 3738-3743.
`Harada, M., Fukasawa, K., Hiraoka, B. Y., Mogi, M., Barth,
`A. & Neubert, K. (1985) Biochem. Biophys. Acta 830, 341-344.
`Kettner, C. A., Bone, R., Agard, D. A. & Bachovchin, W. W.
`(1988) Biochemistry 27, 7682-7688.
`
`Page 4 of 4
`
`Page 4 of 4