Complement-like protein from the phylogenetically primitive vertebrate,Eptatretus Stouti, is a Humoral Opsonin

, pp. 379-384, 1992

HE RATE, ;ONIN*

EMENT-LIKE PROTEIN ] ~IETICALLY PRIMITIVE :S STOUTI, IS A H U M O I RAFTOS,'~ JEFFREY W . H O O K

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ol of Biological and Biomedical Sciences, [ t ' . u . Box 12.~, l~roaoway, N.:S.W. 2007. 200/, Australia. Australm. Tel.: l w~ N.S.W. (02) 33

,~chnology, Sydney, ~2) 330 4095

(Received 5 March 1992; accep ,pied 8 April

Abstract--l. Serum from the Pacific hagfish, Eptatretus stouti, stou contains 2. CLP from unfractionated hagfish serum and from affinity-purifie affi surfaces. 3. Incubation with CLP enhances the phagoeytosis of yeast yq by hag 4. CLP-mediated opsonization can be inhibited by anti-CLP ant antil L(+)rhamnose. 5. Additional opsonic factors are also evident in hagfish ~sh serum.

INTROI~UCTION ,ntibodies and complement proteins are the major numoral umoral opsonins of all vertebrate classes except the le phylogenetically primitive Agnatha (Marchalonis and Schluter, 1989; Farries and Atkinson, 1991). A, number of groups have independently isolated and partially artially characterized humoral molecules from agnath athans which exhibit physicochemical or functional similarities to antibodies (Marchalonis and Cone, 1973 ~73; Marchalonis and Edelman, 1968; Litman et al., 1970 ~70; Hagen et al., 1985; Hanley et al., 1990; Raison el!.a!., 1978a,b; . . . Kobayashi . . .et al., . 1985; . . Varner . et al., 1991). However, recent detailed iled molecular analyses of one such protein do not indicate homology with immunoglobulin (Hanley ett al., 1992). Genomic and eDNA sequencm aencing has shown that a humoral recognition factor from the Pacifc hagfish, Eptatretus stouti, is homologous to mammalian complement proteins rather ;r than immunoglobulin (Hanley et al., 1992). This E. g. stouti complement-like protein (designated CLP) was as originally identified by its induced response to str¢eptococcal immunization (Raison et al., 1978a). CLP has m, has aa molecular weight of approximately 210 kDa and is composed of light and heavy chains (Raison et al.., 1978b). Although CLP heavy chains are of comparable arable molecular weight to those of mammalian IggM, two discrete forms (HI, 77 kDa and H2, 70 kDa )a) are expressed so that either disulphide-linked heterodimq :rodimers (H l-H2) or H 1 homodimers are non-covalenff ntly associated with light Ire protein (Raison et al., (30 kDa) chains in the maturq 1978b; Hanley et al., 1990). md eDNA encoding the Sequencing of genomic and CLP-H1 chain has revealed homology to a family of *This work was supported byy the Australian Research n l ~ 'o^~t,4^~,^.~! it:) . . . . . t . Council. D.A.R. is an Australian Pos Fellow of the Australian Research C tAuthor to whom correspondence shoul

like protein (CLP). binds to yeast cell D(+)mannose and

mam mammalian pr, includes the complement )onents C3, C4 and C5, as well w as sex-limited c°mt prot~ (Sip) and ~2-macroglobulin (Campbell et aL, protein 1988: ~; Farries and Atkinson, 1991; 1991 Hanley et al., 19921 1992). Derived amino acid seque~ uences for CLP-H1 exhit regions with up to 50% identity exhibit id to CA, C5 and Sip. Homology is also reflected :eflected by the apparent intro intron/exon structure of the hagf gfish gene (Hanley et al., 1992). These observations suggest that complementmediated reactions are a predominant humoral de fence mechanism of agnatham aathans. Such a view is cc consistent with evidence from phylogenetically Non-specific more primitive (invertebrate) animals. anita recognition mechanisms have been implicated in the immunological reactions of many invertebrates (Ratcliffe et al., 1985; Raftos, 1992):). Agnathans may be the last animals in evolution to rely on such defence reactions in the absence of a highly specific, adaptive humoral immune respor ~onse characterized by the appearance of rearranging lmmunoglobulins. iJ In this study, we show that CLP iis involved in the defence reactions of agnathans. Complement-like protein from E. stouti is shown hhere to bind and opsonize yeast. MATERIALS AND METH HODS Animals

Pacific hagfish, E. stouti, were purcl ~urchased from Pacific Biomarine Labs, Inc. (Venice, CA). lIn our laboratory, hagfish were maintained at 15°C in 240-1 240-] aquaria filled with aerated, sand-bed filtered sea-water. Media

Sea-water (FSW), that was taken from frov aquaria and then sterilized by filtration (0.45 tam filters, Millipore, 1 Bedford, MA), was used to prepare target a .'t cells and to wash hagfish • tured ~'_ in hag-RPMI culture MI-1640 (908rag/l; Cyto~.ustralia) and penicillin G l--..I

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ontaining the monowere prepared as de1983; Raison and murine monodonal hagfish CLP (Raison was used here as an recognizes human r ) ' e l o l n ~ . ~ l . l l t l g e l l ~ [ D U U A e l U l . , l T O J )3). , Hybridoma l t l ) ' l . ) l l l d I J l I l ( l superoul./~ltants were concentrated 5-fold (Centricon-100, Amicon, tvers, MA) and adjusted to hagfish tonicity by adding 0/~l/ml 20% w/v NaC1.

~gfish leukocytes and serum Blood (1-5 ml/animal) was harvested from the tail sinus anaesthetized (0.075% w/v tricane methane sulphonate; ~na Chemicals, St Louis, MO) hagfish using 2 ml syringes Ied with 26-gauge needles. After centrifugation (200g, nin, 4°C), serum was removed and stored frozen (-20°C). ood ceils were resuspended to twice the original blood lume in 1.2% w/v NaCI buffered with 20 mM sodium osphate (pH 7.4). Two millilitres of these suspensions xe overlaid onto 6 ml Nycodenz (22% v/v in 1.2% w/v IC1 + 20 mM phosphate, pH 7.4; BDH Chemicals, dney, Australia) and centrifuged (800g, 20 rain, 15°C). ukocytes were harvested from Nycodenz gradients, tshed twice through 5 ml ice-cold FSW (200g, 10rain, Wit~lll~)d 4ocC), and resuspended in FSW to 1 x l&cells/ml. For agoeytosis assays, 500 #1 aliquots of these suspensions pha8 w e r)re e added per well to 24-well tissue culture plates (Corning, ;w York, NY). Plates were centrifuged (100g, 5min, New temperature) and 500 #1 bag-KPMI was added per r o o om m dl. Leukocytes were allowed to adhere to culture plates for well. at least 2 h r (15°C, normal atmosphere) prior to use in phagocytosis assays. Purification of hagfish CLP CLP was purified from 5 mlI hagfish serum by affinity previously (Hanley et al., chromatography as described 1990). Serum was diluted 1 : 1 with 0.14 M sodium phostg 0.02% w/v sodium azide phate buffer (pH 8.0) containing and applied to an affinity chromato~ atography column prepared qBr-activated ctivated Sepharose 4B with 4.5 mg H-45 IgG per ml CNBr-activ 's instructions (Pharmacia, according to the manufacturer's ~re eluted and collected in Uppsala, Sweden). Proteins were 0.14 M sodium phosphate buffer: Coil 8.0) containing 0,02% w/v sodium azide. Elution was monitored at 280 rim. Unuted and re-applied to the bound serum proteins were eluted column. Proteins that remainedI unbound after the second .ed serum) were collected in passage (designated CLP-depleted aat had bound to H-45 was a final volume of 50 ml. CLP that 5) and immediately neutraleluted with 0.1 M glycine (pH 2.5) 0). The fraction containing ized with 1 M Tris-HCl (pH 8.0). filtration (Amicon, YM-10) CLP was concentrated by ultrafiltration ~500#g/ml). All fractions and finally adjusted to 10ml (~500 were stored at 4°C.

Binding CLP to ) The ability of h by immunofluore~ (5 x 106) were in 100#l FSW con hagfish serum, affi When required, E prior to mixing ceils were washed 4°C) and incubat H-45 or K-121 h,. anti-mouse IgG-F temperature; CSL twice through l 1 containing 1% w contai: Fluorescence s Dickel Dickenson FACS Sunnyrvale, CA) t at at 400 400 roW. Data sity av averaged fron replica )licates per tre~

bind target cells was tested and flow cytometry. Yeast in, room temperature) in s concentrations of either LP or CLP-depleted serum. ~d to serum or CLP 30 rain s. After incubation, target I ml FSW (16,000 g, 8 sec, om temperature) in 100 #1 rnatants followed by sheep tion in FSW, 60 rain, room ~ed target cells were washed resuspended in 1 ml FSW lehyde. quantified with a Becton meter (Becton Dickenson, ion laser tuned to 488 nm as mean fluorescence intenrescence intensities of three

6sh opsonizat Hagfis a hagfish leukocyte phagoasonic activit' Ops ned yeast ( 5 × 1 0 ~) were cytosis assay. C )rain, room temperature) opsonized by in ous concentrations of unin 100,ul 10£ FSW c airy-purified CLP or CLPfractionated hagf fractic ed, these solutions were depleted serum. ~s carbohydrates (Sigma) or preinc ~reincubated with rior to their addition bridoma supernatants for 30 rain prio hybrid to yeast. After Aft~ opsonization, yeast were washed twice through 1 ml )ended at 5 × 107/ml in g, 8 see, 4°C) and resuspend FSW (16,000 ( lsions were overlaid FSW. Fifty mi~rolitres of these suspens dhered to that had been adhered t 24-well plates as onto leukocytes 1( were rinsed leuk( descril3ed above. Wells containing leukocytes 500 #1 FSW (15°C) which was removed immedionce with v ately prior to the addition of yeast. Phagocytosis was 15 to 180 rain. allowed fi allowe to proceed for periods ranging from Wells were then flushed gently three..times time with 500 #1 FSW flates for 15 rain (4°C) (15°C). (15°C) Leukocytes were fixed to plate,' with ice-cold i being rinsed with methanol (200/~l/well) before b~ )ercentage of leukoextensively with distilled water. The pe yeast cells was cytes that had phagocytozed one or more m( se-contrast, tissue culture determined using a 400 × phase-contr~ were inspected microscope. A minimum of five fields of view x per well ( ~ 50 leukocytes/field of view). Data ] are presented as phagocytic stimulation indexes (PSI) where: yeast PSI = % leukocytes phagocytozing opsonized ( etozing yeast incuper % leukocytes phagocytoz bated with FSW alone.

Statistical analyses Statistical Software, Solo statistical software (BMDP St Inc., Los Angeles, CA) was used for all a statistical calcubetween lations. The statistical significance of differences d tudent's t-tests. Differtrials was determined by two-tailed Stude ences were deemed to be significant for P < 0.05. RESULTS

Target cells

Binding o f C L P to yeast

Saccharomyces cerevisiae (baker's ~ker's yeast) were used as targets for binding or opsonization tion by hagtish CLP. Yeast were grown overnight in YPT media at 37°C (Ausubel et aL, edia at 1989). They were then washed twice thro ;wice through FSW (t000g, 0 s cells/ml in FSW, with or 10 min, 4°C), resuspended to 10 cell,, d, and autoclaved (100°C, without 0.8% w/v Congo Red, washed three times in FSW t 5 rain). Autoclaved yeast were washed (1000 g, 10 min, 4°C), resuspended to 5 stored frozen (-20°C).

Complement-like protein from h~ hagfish serum was capable of binding to yeast cells (Fig. 1). Yeast incubated with affinity-purified CiI P followed by H-45 (anti-CLP) a n d a n t i - I g G G--FFII'Tf C exhibited significantly higher fluorescence t h a n ct:ontrols incubated with H-45 a n d a n t i - I g G - F I T C alone alon ( P < 0.05, 1 : 1 2LP fitted a n asymptotic e maximal (asymptotic)

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Opsonization of yeast by CLP Phagocytosis was enhanced ed by preincubating yeast with afffinity-purified CLP (Fij Fig. 3). The phagocytosis of yeast opsonized by CLP LP increased over time. Approximately 5 times as many hagfish ieukocytes had ingested opsonized tar ~rget cells after 180min (4.2% of all leukocytes) when coml in which leukocytes were exposed t( in FSW (Fig. 3). The phagocyto

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O~)sonization of yeast with CLP was significantly inhibited by co-incubation with 50/~g/ml L(-~-)inhib mnose or D(+)-mannose (Fig. (Fig 4). Nine other rhar~ carbohydrates tested at the same cconcentration did carb( not ,,significantly alter phagocytic activity (P > 0.05 Fig. 4). opsonized without carbohydrate, carbo vs yeast y~ ~reincubating yeast ~sonization was abrogated by pr Opsc (Fig. 5, P > 0.05 vs with CLP and > l0 mM mannose (l: unol:~sonized controls). Rhamnose reduced, but did not qeliminate, opsonic activity at any of the cont.05 vs unopsonized centrations tested (Fig. 5, P < 0.0~ centr controls). Opsonic activity was also inhibited inhibit~ by preincubating yeast with CLP in the presence esence of ( EDTA (Fig. 6). at 20mM Half-maximal inhibiton was evident evi eliminated EDTA. Concentrations />50 mM EDTA I opsonic activity (P > 0.05 vs unop,~sonized controls).

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EDTA inhibits binding Preincubation of affinity-l:purified CLP with EDTA decreased its capacity to bind nd yeast (Fig. 2). Significant inhibition of H-45-s -specific immunofluorescence was evident at 5 mMl EDTA (P < 0.05 vs no EDTA). Specific binding was vas eliminated by EDTA concentrations > 50 mM (P >0.05 vs unstained controls).

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vel of fluorescence among CLP-incubated yeast was ?proximately 6.5 times greater than that of unained controls without CLP (Fig. 1). The specificity f H-45 for CLP was confirmed by the absence of ~hanced fluorescence of yeast incubated with CLP ad then immunostained with an irrelevant antibody and (-121) and anti-IgG-FITC (P >0.05 vs controls (K-121) with no CLP). H-45-specific immunofluorescence was also evident when yeast were incubated with unfractionated haagfish serum (Fig. 1). The maximal (asymptotic) ahancement of fluorescence afforded by incubation enhancement 'ith serum was approximately 4 times greater than with 3ntrols without serum (P < 0.05, 1 : 1 dilution), controls ~gain, immunostaining Ag tg of serum-incubated yeast 'ITC did not significantly with K-121 and anti-IgG-FITC controls trois (P > 0.05, data alter fluorescence relative to) control not shown). Incubation of yeast with CLP-depleted serum fol-FITC did not enhance lowed by H-45 and IgG-FITC fluorescence intensity whent compared to either unstained or K-121 immunostained :ainedt controls cc (P > 0.05, Fig. 1).

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ig. 4. Phagocytic stimulation indexes (PSIs) for hagfish ukocytes overlaid for 30 rain with yeast that had been )-incubated with afffinity-purified CLP (1:5 in FSW) and )mM of various carbohydrates. Carbohydrates marked ith an asterisk significantly reduced PSIs relative to yeast cubated with CLP alone (P < 0.05). Bars = SEM, N = 3.

~psonization by serum and CLP-depleted serum Opsonization of yeast with serum and CLPepleted serum enhanced phagocytosis relative to ntreated controls (Fig. 7). Dose-responses for serum umreateu nd CLP-de CLP-depleted serum were asymptotic. The maxiand ml PSIs for yeast opsonized by serum and CLPreal deepleted serum were approximately 14.5 and 4.6, r e s,~spectively. Opsonization with afffinity-purified CLP ,~sulted in a maximal (asymptotic) phagocytic acresulted vity that was significantly lower than that for serumtivit, opsonized psonized yeast (P < 0.05, 1 : 1 dilution, PSI ~ 7.5).

Inhi ~hibition o f opsonization with anti-CLP antibody .5) inhibited the opsomzAnti-CLP antibody (H-45) g. 8A). Preincubation of ation of yeast by CLP (Fig. yeast with CLP and up too 1:5 dilutions of H-45 ,ted opsonization relative culture supernatant eliminate, to unopsonized controls (PP > 0.05). H-45 also decreased the ability of unfractionated :tionated serum to opson-

Fig. 66. Phagocyti that had 1~ been ov incubated incub~ in affin concentrati

adexes (PSIs) for leukocytes tin with yeast that had been ,P (1:5 in FSW) and various Bars = SEM, N = 3.

(P < l opsonized without H-45, ize yeast y~ 1:5 dilution). erum retained approximate mately 50% o : activity at the highest cono:entration ~ :1 (1 : 1 dilution, P < 0.05 -1-45 did not inhibit upvs unopsonizec m sonization by CLP-depleted serur serum at any of the soniz concentrations tested (P > 0.05 vs yeast y opsonized in curio the absence of H-45). Co-incubation of yeast with CLP, CL[ serum or CLPCc 31eted serum in the presence of an a irrelevant antidepk body body (K-121) did not significantly alter phagocytic activity when compared to yeast opsonized in the activ absence of K-121 (P < 0.05, Fig. 88B). abse] DISCUSSION

yeast. The Hagfish CLP binds to and opsonizes ups( was demoncapacity of CLP to bind target cells ( strated in an immunofluorescence assay using the )ecificity of this detecanti-CLP antibody H-45. The specif tion system is confirmed by the 1lack of enhanced .... -=---. 20

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timulation indexes (PSls) of Fig. 5. Effect on phagocytic stimulatio ~nltv.n]=rifipd ("T P (1 .=[ i n co-incubating yeast with affinity-puri FSW) and various concentrations of t.(+)-rhamnose. Bars = SEM

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fluorescence staining in co ntrols from which CLP vhich irrelevant antibody was omitted and those in which (K-121) was substituted forr H-45. Moreover, H-45specific immunostaining was+not evident among yeast md been depleted of CLP incubated with serum that had by affinity chromatography. The binding of CLP to yeast facilitates phagocytosis by hagfish leukocy tes. Incubation of yeast with affinity-purified CLP significantly enhances This increased phagocytic their subsequent ingestion. This tble to the interaction of activity is directly attributable yeast and CLP. Anti-CLPP antibody inhibits the ated with affinity-purified phagocytosis of yeast incubatec CLP. Irrelevant antibody (K-121) has no such inhibitory effect. These data suggest that CLP acts as an opsonin tosis by coating foreign and so promotes phagocytosis ween the binding of CLP particles. A correlation between to yeast and the stimulationn of phagocytosis is indicated by the sensitivity of' CLP to inhibition with EDTA• EDTA inhibits the binding of CLP to yeast surfaces and decreases thee phagocytosis of CLPincubated yeast. This suggests tt opsonization are concomitant, dependent functions.

Iso inhibited by selected Opsonization )anel of 11 sugars, Lcarbohydrates. rhamnose and significantly decrease • Such inhibition has a the opsonic act P may act as a divalent number of expl ate-specific lectin. Mancation-dependel inhibit recognition by nose and rhan e ligands on the surface competing with tural similarity of rhamof yeast cells. T] vary by only a single nose and man~ aent with such discrimimethyl/ethyl gr( )n. Carbohydrate inhinative, lectin-li ; i t i o ; spectra : ase for CLP have been bition aine the carbohydrate used frequentl ,arious species (Ratcliffe ~cificities of 1 specit e t al.,,1985; Sh y is inconsistent with the However, lecl Ho ,tween CLP and compevolu evolutionary re bears homology to the lemer :ment compot maml mponents, C3 and C4, mammalian co bitor, ~t2-macroglobulin as well w~ as the these mammalian pro(Hanl (Hanley et al., s which covalently interthi teins possess i determinants on target act with hydro or hydrolytic activation ~lecules after mole~ )shita, 1991; Tack et al., (Campbell (Cam et aJ evidence suggests that 1 ;'l t.;1~, l ~ l ~ 1 . . , g ; l l L g; ¥ IU.E~ll 1l ~~.O J ) , 1980; Tack, nathans, including comp dement-like proteins from agn~ thiolester CLP, also contain highly reactive r E. stouti stt et al., 1984; ;roups (Hanley et al., 1992; Nonaka Nora groul Osterberg et al., 1991). Hence, it is likely that CLP Ostet )osure and covalent ,inds to target cells via the expom binds bindi binding of thiolesters. The ability of mannose and rham diated opsonization may ~mnose to inhibit CLP-mediated c reflec a degree of selectivity in the ligands which are reflect 1 boun by thiolesters. In mammals, small variations bound among amor C4 isotypes allow them to discriminate berminants on target tween tweel hydroxyl and amino determ imilar selectivity is cells (Yu et al•, 1986). Perhaps sirr raze carbohydrates employed in agnathans to recogni on pathogenic targets. While covalent binding is one plausible explanation for the action of CLP, there is no evidence to suggest that binding results from aactivation of CLP lar to that by a proteolytic cascade similar th of mammalian ~sonize yeast in the complement. CLP can bind and ops rots. The maximum absence of other serum component with phagocytic activity arising from opsonization o roximately twice the level unfractionated serum is approximat ~sonization with affinitythat can be obtained by opsonizat purified CLP in the absence of oother serum components. However, this additional opsonic activity ttributed to non-CLP of unfractionated serum is attribt >sonic activity reopsonic molecules. Significant op,, mained in serum that had been depleted of CLP the opsonic by affinity chromatography• Moreover, More~ activity of unfractionated serum was only partially inhibited by anti-CLP antibody• These data suggest that CLP does not require They proteolytic activation by serum components. cc ~dditional opsonic factors also reveal the existence of addition in the serum. It is impossible to assess the relative )somns to phagocytosis contribution made by these opsonir Jdy confirms that CLP ve reactivity of hagfish

DAVID A. RAFrOS et al.

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)h T., Natsuume-Sakai S., and Takahashi M. (1984) nplement component homont of the mammalian comp133, 3242-3249. and Ikai A. (1991) X-ray )rotease inhibitor, a protein ~ment and ~2-macroglobulin.

Biochemistry "~

Raftos D. A. (1! systems in in (Edited by Co University Pre Raise R. L. anc Raison bet bearing blood

ent of primitive recognition 1 Development Immunology

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