Antiflammins: Bioactive Peptides Derived from Uteroglobin

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Antiflammins

Bioactive Peptides Derived from Uteroglobin

LUCIO MIELEa

Cancer Immunology Program, Cardinal Bernardin Cancer Center,Loyola University Chicago, Maywood, Illinois 60153, USA

ABSTRACT: Uteroglobin/Clara cell 10-kDa protein (UG/CC10) is a hormonallyregulated small secretory protein that has a variety of in vitro and in vivo phar-macological effects. These include a potent anti-inflammatory activity andinhibitory effects on neutrophil migration, thrombin-induced platelet aggrega-tion, in vitro chemoinvasion, as well as “tumor suppressor”–like effects andother properties. Several mechanisms of action have been proposed for theseeffects. Pharmacological properties suggest that UG itself or substances de-rived from it may be used as experimental drugs for several indications. Thegroup of oligopeptides collectively known as “antiflammins” (AFs) were origi-nally described in 1988. Their design was derived from the region of highestsequence similarity between UG and another group of proteins with anti-inflammatory properties, the lipocortins or annexins. Nanomolar concentra-tions of these peptides can reproduce several of the pharmacological activitiesof UG, including its in vivo anti-inflammatory effects and inhibition of plateletaggregation. The AFs have been safely and effectively used to suppress inflam-mation and fibrosis in several animal models. Progress in clarifying the mech-anism of action of the AFs may facilitate the structure-based design of a novelclass of potent anti-inflammatory, antichemotactic drugs.

BIOLOGICAL ACTIVITIES OF UTEROGLOBIN FAMILY PROTEINS

For over three decades, the proteins of the uteroglobin (UG)/Clara cell “10”-kDa(CC10) family (henceforth, UG) have presented a fascinating puzzle to the scores ofinvestigators who have studied them. UG proteins are a highly conserved family ofsmall-molecular-weight homodimers that are secreted by mucosal epithelia in theairways and reproductive tract and by numerous other tissues. UG expression is reg-ulated by progestins, glucocorticoids, androgens, prolactin, and most likely otherhormones and cytokines.1,2 Despite their small size and extensive structural, biolog-ical, and genetic characterization, our understanding of the physiological role(s) ofthese proteins remains incomplete. This is largely due to the fact that UG proteinshave a bewildering array of in vitro and in vivo biological activities, in sharp contrastwith their deceptively simple structure.1,2 These include in vitro inhibitory effects onplatelet aggregation3 and on neutrophil chemotaxis,4,5 in vivo inhibition ofcarrageenan-induced acute inflammation,6 inhibition of chemoinvasion by various

aAddress for correspondence: Cancer Immunology Program, Cardinal Bernardin Cancer Cen-ter, Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153. Voice: 708-327-3362; fax: 708-327-3238.

[email protected]

129MIELE: ANTIFLAMMINS

transformed cell lines,7–9 and inhibition of anchorage-independent growth of trans-formed cell lines.10,11 It is still unclear which of these multiple activities are physio-logically relevant and which can be considered pharmacological effects. Targeteddisruption of the mouse UG gene causes severe inflammatory nephropathy withfibronectin/IgA deposits12–14 and increases the incidence of malignancies.11 A dif-ferent model of mouse UG gene inactivation shows increased sensitivity to oxidizinginsults to the lung.15

To make matters even more complicated, no single mechanism has emerged sofar that can explain all the biological activites of UG proteins. Their biochemicalproperties described so far are almost as numerous as their biological effects andinclude, among others, the following: (1) binding a variety of small hydrophobicmolecules such as progestins, retinoids, and toxic xenobiotics;16–21 (2) inhibition ofsecretory phospholipases A2 (sPLA2’s);6,22–26 (3) binding to a saturable membranereceptor;8,9 and (4) binding to fibronectin.12–14 Ca2+-dependent binding to nega-tively charged liposomes27 and specific binding to a microsomal protein, which mayor may not be related to the membrane receptor,28 have been reported as well.

The relationship between biochemical and biological properties of UG proteinsis complex and incompletely understood. The antichemoinvasion effect appears tobe mediated by the UG receptor,8,9 while fibronectin binding is likely to participatein the prevention of IgA nephropathy.12–14 The mechanisms of the in vivo anti-inflammatory and tumor-suppressive activities and the possible relationship betweenthese two properties are less clear. In principle, inhibition of sPLA2’s, the UG recep-tor, and fibronectin binding may all participate in these effects and it is not possibleto rule out additional, as yet undiscovered, mechanisms.

STRUCTURE-FUNCTION RELATIONSHIP OF UG PROTEINS:THE ANTIFLAMMIN PEPTIDES

Aside from the thorny question of physiological functions, it is apparent from thisbrief summary that the pharmacological activities of UG proteins make them ex-tremely attractive candidates for therapeutic applications. Recombinant human UGis an excellent drug candidate in its own right because of its small size and excep-tional stability29 and because it can be produced in bacteria in its native, disulfide-bonded form using an expression system that we developed for that purpose30,31 andsubsequently improved for industrial use.32 Additionally, understanding the struc-tural basis for UG’s pharmacological activities could open the way to the develop-ment of orally active, nonpeptide drugs that mimic UG activities. Based on theknown information on the biological activities of UG, possible indications for UG-mimetic drugs range from inflammatory and autoimmune disorders to lung and pros-tate cancers.

In 1988, we6 identified a 9-amino-acid region within α-helix-3 of rabbit UG(residues 39–47) that could reproduce the anti-inflammatory activity of the purifiedparent protein in vivo. This led to the development of a family of pharmacologicallyactive oligopeptides collectively known as “antiflammins” (AFs) (reviewed in refer-ence 33). These peptides were derived from the region of highest sequence similaritybetween UG and annexin I/lipocortin I, another glucocorticoid-induced protein orig-

130 ANNALS NEW YORK ACADEMY OF SCIENCES

inally identified because of its anti-inflammatory properties.6 Over the past decade,the pharmacology of AFs has been explored by numerous groups and these peptideshave clearly emerged as potentially useful therapeutic agents. Several AF peptidederivatives have been designed, additional pharmacological activities have beendescribed, and mechanisms responsible for inactivation of AFs have been identified.The structures of AF peptides described to date and the putative structure of a humanUG-derived antiflammin are shown in TABLE 1, together with a proposed nomencla-ture for this group of peptides. The consensus sequence shows a specific pattern ofhydrophobic and hydrophilic residues that can be modeled as an amphipathic α-helix, with invariant Lys and Asp residues, respectively, at 4 residues and 1 residuebefore the C-terminus. These correspond to invariant Lys 43 and Asp 46 in thesequence of UG.

PHARMACOLOGICAL PROPERTIES OF AFS

In our original report, AFs, purified rabbit UG, and recombinant annexin/lipo-cortin I potently inhibited carrageenan-induced rat-paw acute inflammation.6 Theireffect was comparable to that of dexamethasone (10 µg/kg) and superior to that ofindomethacin (1 mg/kg). Histologically, the most striking features were a dramaticdecrease in edema and in the number of neutrophils infiltrating carrageenan-injectedareas. Subsequently, Vostal et al. showed that AFs have a weak inhibitory effect onADP-induced platelet aggregation in vitro.33 Camussi et al.34–36 demonstrated thatAFs inhibit the synthesis of platelet-activating factor (PAF) induced by TNF or byphagocytosis in rat macrophages and human neutrophils or by thrombin in endothe-lial cells. PLA2 activity in cell lysates and arachidonic acid (AA) release were inhib-ited by AFs, as was the activation (but not the catalytic activity) of acetyl-CoA-lyso-PAF acetyltransferase in intact cells. IC50 values for these activities were between50 and 100 nM. Importantly, pretreatment with AFs before cell activation was moreeffective than treatment after application of the stimulus. These same authors also

TABLE 1. Sequence of AF peptides and proposed nomenclature

Name Sequence Parent protein Reference

AF-1 MQMKKVLDS Rabbit UG 6

AF-2 HDMNKVLDL Annexin I 6

AF-2A HDANKVLDL Annexin I 34

AF-2Na HD(nL)NKVLDL Annexin I 34

AF-2NSa HD(nL)NKVLDS Annexin I 34

AF-3 SHLRKVFDK Annexin V 35

AF-4 LRKVFDK Annexin V 35

AF-5 AQLKKLVDT Human UG This article

Consensusb XXX-FIL-FOB-FIL-LYS-LEU/VAL-VAL/LEU/PHE-ASP-FIL

anL = norleucine.bXXX = indetermined; FOB = hydrophobic; FIL = hydrophilic.


demonstrated that AFs inhibit neutrophil chemotaxis and aggregation and blockintradermal Arthus reaction induced by injection of TNF or C5a in rats.34–36 They37

also showed that AFs inhibit TNF-induced cytoskeletal reorganization in human en-dothelial cells. Subsequently, Ialenti et al.38,39 showed that AFs and annexins/lipo-cortins inhibit the late phase of carrageenan-induced rat-paw edema, which ismediated by eicosanoid release, but not the early phase, which is primarily mediatedby vasoactive amines. Chan et al.40 showed that AFs are as potent as dexamethasonein the treatment of experimental endotoxin-induced uveitis, a model of autoimmuneanterior uveitis. Reduced PLA2 activity in the aqueous humor was documented.Perretti et al.41,42 described a novel AF peptide derived from a region of annexin/lipocortin V that is homologous to the “AF” region of rabbit UG. This peptide andthe lipocortin I–derived AF-2 inhibited the contractions of isolated rat stomach stripsinduced by the application of porcine pancreatic PLA2. These AFs also inhibitedprostaglandin E2 production induced by bradykinin in human skin fibroblasts or byopsonized zymosan in rat peritoneal macrophages. Sierra-Honigmann and Murphy43

showed that recombinant lipocortin I and AF-2 inhibit IL-1-mediated activation ofmurine Th2 cells in vitro. Lloret and Moreno44 showed that AFs inhibit collagen-induced platelet activation and that they have a potent anti-inflammatory effect bylocal and systemic administration in carrageenan- and phorbol ester–inducedinflammation, but not when inflammation was induced by treatment with AA orsnake venom (Naja naja) sPLA2. At the same time, Calderaro et al.45 showed thatAFs inhibit PGE2 production and chloride secretion induced by Ca2+ ionophoreA23187 in rabbit distal colonic mucosa. AA-induced PGE2 production was inhibitedby 100 nM AF-2 to the same extent as 10 µM indomethacin. Chloride secretion andcAMP accumulation induced by direct administration of PGE2 were not inhibited,suggesting that, in this system, AFs inhibit PG production. A direct inhibitory effecton colonic mucosa cyclooxygenase with 100 nM AF-2 was observed by theseauthors. On the other hand, Lloret and Moreno46 reported that AF-1 and -2 signifi-cantly inhibited carrageenan-induced inflammation, mast-cell degranulation, leuko-cyte infiltration, and histamine release in rat paws, but did not inhibit edema causedby snake venom (Naja naja) sPLA2. Cabre et al.47 showed that AFs inhibit inflam-mation in vivo caused by carrageenan, croton oil, and oxazolone, but not snakevenom (Naja naja) sPLA2. Human synovial (group II) sPLA2 was not inhibited byAFs when bacterial cells were used as the substrate, but was inhibited in a mixedmicellar assay. Subsequently, the same group48 showed that topical treatment withAFs have a potent and dose-dependent inhibitory effect on edema, cell infiltration,plasma leakage, and leukotriene B4 (LTB4) production in phorbol ester–treatedmurine ears. Edema induced by topical administration of AA was not inhibited, sug-gesting that the AFs act in this model by inhibiting either AA release or the lipo-oxygenase pathway. In a different in vitro model of murine 3T6 fibroblasts orperitoneal macrophages, the same authors49 did not observe effects of AF-2 on eitherAA release or metabolism, while a potent effect on leukocyte migration was ob-served. The authors suggest that this effect may be due to inhibition of leukocyte ad-hesion to endothelial cells and that it may participate in the in vivo anti-inflammatoryeffects of the AFs. More recently, Rodgers et al.50 demonstrated that postoperativeadministration of AF-2 by mini-osmotic pumps significantly reduced peritoneal ad-hesions in two different animal models. Peritoneal adhesions are a consequence of


inflammation induced by surgical injury and a significant source of postoperativemorbidity and mortality. These data suggest that AFs are potential candidates for theprevention of this serious postoperative complication. Rayner and Van51 reportedthat AF-2 strongly inhibited substance-P-induced lymphatic vessel contractions, buthad no effect when a thromboxane A2 analogue was used as the stimulus, suggestingthat AF-2 acts upstream of AA metabolism in this model. Mize et al.52 used AF-1 ina chlorpromazine-induced skin inflammation model and showed that this peptidecan be delivered intradermally by iontophoresis, retaining its anti-inflammatoryactivity as determined by inhibition of erythema and lesion formation. This suggeststhat AFs may be used for local treatment in dermatological indications. PGE2 pro-duction was not inhibited in this model, in contrast with the results of Perretti et al.in human fibroblasts and murine macrophages41,42 and of Calderaro et al. in rabbitcolonic mucosa45 (see earlier). In an animal model of conjunctivitis, Li et al.53 dem-onstrated that topical administration of AF-2 inhibits ocular inflammation caused bycompound 48/80. Interestingly, the expression of sPLA2’s and that of inducible NOsynthase (iNOS) were inhibited by AF-2 and by dexamethasone in this model. Gaoet al.54 have recently shown that 1 nM AF-1 inhibits lymphatic vessel contractioninduced by ATP. The latter is thought to be mediated by increased expression ofsPLA2. Very recently, the effects of AF-1 and -2 on leukocyte adhesion were studiedin detail by Zouki et al.55 These authors demonstrated that AFs and recombinantlipocortin I inhibit the changes in L-selectin and CD11/CD18 induced in human neu-trophils by PAF and IL-8. The peptides and the recombinant protein had similardose-response relationships. Moreover, AFs inhibited adhesion of human neutro-phils to human coronary artery endothelial cells activated by LPS. This effect wasadditive with anti-E-selectin and anti-L-selectin antibodies, but not with anti-CD18antibodies. The authors conclude that AFs modulate human neutrophil adhesionprimarily by affecting CD18 expression and that these peptides are potential candi-dates for therapeutic indications that require modulating human neutrophil adhesionin vivo.

In summary, data from numerous groups have shown that AFs are highly potentanti-inflammatory agents that reproduce many pharmacological activities of theirparent proteins, UG and annexins/lipocortins. Together, these data show that AFs:

(1) are active in vivo by parenteral (intradermal and systemic) and topicaladministration and can be delivered by iontophoresis and microosmoticpumps;

(2) have highly potent anti-inflammatory activity in diverse animal models invarious organs and tissues, and prevent postoperative surgical adhesions;

(3) inhibit leukocyte adhesion and function, modulate the expression of adhe-sion molecules on neutrophils, and inhibit mast-cell degranulation, smoothmuscle contraction, and lymphatic vessel contraction;

(4) inhibit the production or release of inflammatory mediators, includingPAF, eicosanoids, and histamine, and possibly modulate the expression ofsPLA2’s and iNOS;

(5) have variable effects on AA release and the cyclooxygenase or lipooxygen-ase pathways, depending upon the experimental model.

These data indicate that these peptides, or structural analogues designed fromthem, are potentially attractive candidate drugs for a variety of anti-inflammatory


uses, including dermatological and ophthalmological applications, and for the pre-vention of postoperative adhesions. The effect of AFs on neutrophil adhesion tohuman coronary endothelial cells suggests that they may be useful for such indica-tions as prevention of reperfusion injury in myocardial ischemia and infarction. Al-though a formal toxicity study has not been performed, it is important to note that noapparent in vivo toxic effects have been reported thus far when AFs were tested atdoses as high as 2 mg/kg in numerous animal models.

MECHANISM OF ACTION OF AFS

As is the case for most drugs and for UG proteins themselves, the multitude of invivo activities of AFs are difficult to integrate into a straightforward model based ona simple mechanism of action. The information obtained to date is summarized be-low. The parent proteins, UG and annexins/lipocortins, inhibit the catalytic activityof sPLA2’s in vitro. The mechanism(s) of this effect is still the subject of much con-troversy, which cannot be discussed in detail here for reasons of space (for reviews,see references 1, 26, and 33). Briefly, substrate sequestration due to Ca2+-dependentbinding to anionic phospholipids has been proposed as a mechanism of inhibition forannexins,56,57 but others working with different models have questioned this conclu-sion.58,59 Annexins also inhibit intracellular, cytosolic PLA2 activity,60,61 while noevidence of such intracellular activity has yet been described for UG. Similar toannexins, Ca2+-dependent binding to anionic phospholipids has been described forUG.27 However, UG also inhibits sPLA2 in the absence of anionic phospholipidswith phosphatidylcholine vesicles as the substrate.22 Based upon the structural6,62

and sequence similarity25 between UG and sPLA2’s, we originally proposed that UGproteins may interfere with the process of interfacial activation or with the inter-action of sPLA2 with lipid-water interfaces rather than the actual catalytic pro-cess.1,6,24,25,63 Interestingly, UG has been suggested to have a Ca2+-binding loopsimilar to that of sPLA2’s.64 It has been suggested that Asp 46, an invariant residuein the AF region of UG and in AF peptides, is a critical residue in this loop. Thesedata are consistent with our hypothesis that UGs are structural analogues of sPLA2’sand further identify the AF region as an area of high structural similarity betweenthese protein families. AF peptides inhibit porcine pancreatic sPLA2 with micellarsubstrates6,25 and human group II sPLA2 with micellar substrates,47 as well ashuman neutrophil sPLA2,35,36 but like UG they have no effect when bacterial cellsare used as the substrate or with snake venom sPLA2’s (see earlier). Preincubationof pancreatic PLA2 with AF-1 blocks its activity on mast-cell degranulation.65 AFsinteract with lipid-water interfaces66 and inhibit the increase in fluorescence of Trp 3in porcine pancreatic sPLA2 that is normally triggered by binding to micellar sub-strates.25 All in all, the most likely explanation for these observations is that, likeUG, AFs also inhibit either the interfacial activation or the interaction of sPLA2’swith lipid-water interfaces. PLA2 inhibition by AFs had been questioned earlier.67,68

However, in these studies, the peptides also showed no pharmacologic activity,67

calling into question the quality of the peptide preparation used, or were used underdifferent conditions than we used and/or with bacterial cells as the substrate.68 Sincedetails of peptide handling and storage were not discussed in these reports, one pos-


sible interpretation of such findings is that these authors may have been workingwith inactivated peptides (see below). Very recently, Chowdhury et al. (this volume),using site-directed mutagenesis, have definitively confirmed that the C-terminal halfof α-helix-3 is the active site of UG for PLA2 inhibition, as we originally proposed.6

The interpretation of in vitro PLA2 inhibition studies vis-à-vis the mechanism ofpharmacological activity of AFs is complicated by several factors. First, most of thestudies reported so far, with the notable exception of Camussi et al. (see above), havenot used physiologically relevant PLA2’s or substrates, relying instead on commer-cially available digestive or snake venom enzymes and artificial substrates. The re-cent isolation of several mammalian sPLA2’s with structural similarities, but distinctcatalytic properties,69–71 and observations indicating that endogenous mammalianenzymes have vastly different biological activities in mammalian cells compared tosnake venom enzymes72 suggest caution in interpreting data obtained with experi-mental systems of questionable physiologic relevance. In general, the very nature ofPLA2 catalysis makes most assay systems highly artificial and notoriously difficultto standardize. This is especially true with nonlipid-soluble, noncompetitive inhibi-tors such as proteins or peptides. Thus, the role of PLA2 inhibition as a potential invivo mechanism of action of UG and AFs should be studied using mammalian non-digestive enzymes and physiologically relevant substrates. It is likely that differentmammalian enzymes will show different sensitivity to inhibition, depending upontheir affinities for lipid-water interfaces and mechanisms of interfacial activation,and that the type of substrate used will affect the results. The in vivo evidence avail-able to date indicates that UG-deficient mice have higher plasma PLA2 activity.12

A second potential source of confusion in interpreting AF studies is peptideinactivation. In our original studies,6,25 AF peptides were stored in sealed glass vialsunder rigorously anhydrous conditions. Solutions were prepared with ice-cold buff-ers immediately before use and discarded after use. No AF solutions were stored.We25,63 and others34–36,73 discovered that AFs were rapidly inactivated upon storagein solution or under nonanhydrous conditions. Camussi et al.34–36,73 found that AFscontaining Met residues were rapidly oxidized in the presence of air and that reduc-ing agents could protect these peptides from this mechanism of inactivation. Thisproblem was circumvented by Tetta et al.73 by replacing Met with Ala or Nle resi-dues. Additionally, these authors also discovered that AFs were completely inacti-vated by freezing and thawing in solution. This may be a consequence of aggregationand/or precipitation and could be partially reversed by heating solutions at 45°C.More recently, Wolfe et al.74,75 demonstrated that AF-2 spontaneously hydrolyzesin aqueous solutions with pseudo-first-order kinetics. The primary hydrolysis pointwas at the C-terminal side of Asp 46. Under similar conditions, Met sulfoxide wasthe primary oxidation product.76 In summary, there are at least three different mech-anisms through which AF solutions can lose activity upon storage. One of these(freezing-induced inactivation) is not associated with chemical degradation andwould not necessarily be detected by chemical methods such as mass spectrometryor HPLC. This suggests that special attention should be paid to peptide handling inAF studies and that inconsistent results are likely to ensue if this is not done.

A third possible confounding factor in interpreting pharmacological studies withAFs is that not all the in vivo activities of sPLA2’s are mediated by their catalyticactivity. High-affinity membrane receptors for sPLA2’s have been isolated in recent


years.77–83 These receptors belong to the same superfamily as mannose-6-phosphatereceptors (M-type receptors). M-type receptors are multifunctional molecules thatbind PLA2’s via their carbohydrate recognition domains (CRDs) and have an N-terminal fibronectin type II repeat that most likely mediates cell adhesion.82 M-typesPLA2 receptors mediate a variety of cellular effects that had been previously attrib-uted to sPLA2 catalysis. These include stimulation of AA release and eicosanoidproduction from intact cells,84,85 chemotaxis and chemoinvasion,86 smooth musclecontraction,87 and cell proliferation.77 Through M-type receptors, group I sPLA2’sincrease the expression and secretion of group II sPLA2.88 Interestingly, many ofthese processes are inhibited by UG and/or AF peptides in various experimentalmodels, including models in which pancreatic PLA2 is used as the stimulus (see ear-lier). On the other hand, snake venom enzymes, which can readily hydrolyze mam-malian cell membranes, do not appear to respond to AFs in vivo. These observationsraise the question of whether AFs and/or UG may act in vivo on sPLA2 M-typereceptors. Theoretically, there are two different possible mechanisms for such bind-ing: AFs and/or UG may mimic the sPLA2 Ca2+-binding loop,64 which is essentialfor PLA2 receptor binding,89 and thus compete with PLA2’s for the CRD; alterna-tively, UG or AFs may bind the N-terminal fibronectin repeat, without competingwith the PLA2-binding site, and through this they could affect cell adhesion, triggerreceptor internalization,90,91 and thus indirectly prevent PLA2 receptor–mediatedeffects and downregulate the expression of group II sPLA2. UG does bind fibro-nectin,12 but it is still unknown whether AFs do. This possible additional mechanismof action is at present entirely speculative. However, it is consistent with all the avail-able evidence and could help explain several biological activities of UG and AFs, aswell as the variable results on eicosanoid metabolism observed with AFs in differentmodels. Yet another possibility is that AFs may interact with the UG receptor;8,9

whether the latter receptor is related to M-type sPLA2 receptors is still unclear.In summary, AFs inhibit various mammalian sPLA2’s in vitro with micellar sub-

strates, including sPLA2 activity in human neutrophil lysates, and block certaineffects of group I (pancreatic) mammalian sPLA2 on intact cells and organ prepara-tions. AFs do not inhibit snake venom enzymes in vitro nor block their toxic effectsin vivo. Some of the effects that are inhibited by AFs are mediated by the group IPLA2 M-type receptor, raising the possibility that, in vivo, AFs and UG may alsointeract with these PLA2 receptors, either at the same site bound by PLA2’s or at adifferent site. At present, it is impossible to rule out that other mechanisms that havebeen proposed, such as inhibition of PAF synthesis or of cyclooxygenase, and inter-action with the UG receptor, as well as hitherto undiscovered mechanisms, may alsoparticipate in the in vivo pharmacological activities of the AFs.

CONCLUSIONS AND FUTURE DIRECTIONS

Results obtained by many independent groups over the past 11 years have dem-onstrated that AF oligopeptides derived from the C-terminal half of α-helix-3 of UGand from the homologous regions of annexins/lipocortins are extremely active anti-inflammatory agents and reproduce many of the pharmacological activities of theparent proteins. AFs inhibit inflammation in vivo in numerous models, as well as


surgical adhesion formation, smooth muscle contraction, lymphatic vessel contrac-tion, and leukocyte adhesion and activation. Invariant residues in these peptides areLys 5 and Asp 8 (corresponding to Lys 43 and Asp 46 in intact UG). Site-directedmutagenesis confirms that AFs correspond to the active site for this activity of UG.PLA2 inhibition and possibly other activities such as PLA2 receptor antagonism,cyclooxygenase inhibition, and as yet undiscovered mechanisms may contribute tothe pharmacologic activities of the AFs.

A number of critical questions require further investigation: Do the AFs share theeffects of UG on neoplastic cell adhesion, anchorage-independent growth, andchemoinvasion? Is PLA2 receptor antagonism an in vivo mechanism of action of theAFs? Do AFs bind M-type sPLA2 receptors and, if so, do they bind the CRDs or theN-terminal fibronectin repeat? Do they bind fibronectin or other fibronectin repeat–containing proteins and/or the UG receptor? Answering these questions will betterdelineate the spectrum of candidate therapeutic indications for AFs or their deriva-tives and will provide information on possible docking sites for AFs that may beused in the rational design of nonpeptide analogues.

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Antiflammins: Bioactive Peptides Derived from Uteroglobin