Proteins of Crustacean Exoskeletons: I. Similarities and Differences among Proteins of the Four Exoskeletal Layers of Four Brachyurans

15 Pages·1991·6 MB·English

by  J J O'brien

Reference: liiol Bull 181: 427-441. (December, 1991)
Proteins of Crustacean Exoskeletons: I. Similarities
and Differences among Proteins of the Four
Exoskeletal Layers of Four Brachyurans
JACK J. O'BRIEN,1 S. SINDHU KUMARI, AND DOROTHY M. SKINNER2
BiologyDivision, P. O. Box 2009, Oak Ridge NationalLaboratory, Oak Ridge, Tennessee
37831-8077 and East Tennessee State University. Johnson City, Tennessee 37614
Abstract. Mostoftheproteinsextractedfromexocuticle, proteinsofthesame sizeseparatedbydifferencesincharge
endocuticle, and membranous layer of four species of during isoelectric focusing.
anecdysial (intermolt) crabs (the Bermuda land crab Ge-
carcinus lateralis, the rock crab Cancer antennarius, the Introduction
shield-backed kelpcrabPugettiaproducta, andthe south- Arthropods grow by molting, a programmed process
ern shield-backed kelpcrab Taliepusnitttalli)were 31 kDa of developmental changes that occurs prior to ecdysis
or smaller; proteins of similar Mr were common to all (proecdysis) and in the period immediately following ec-
three layers. Proteins from the membranous layer were dysis (metecdysis). After the events of metecdysis are
qualitatively indistinguishable in all four species. More completed, themoltingprocessisconsideredtobefinished
proteins 31 kDa or smaller were similar in size and pi to and the animal enters anecdysis, the intermolt period
proteins from otherexoskeletal layers than were proteins during which there is no further increase in external di-
largerthan 31 kDa. Proteinsextracted fromtheepicuticle mensions. Duringeach proecdysial period, theepidermis
ofG. lateralis included a group offive ranging from 54 underlying the exoskeleton synthesizes a new epicuticle
to 42 kDa that bound 45Ca++ in vitro. The group was not and exocuticle, the two outermost layers ofthe new exo-
seen in other layers ofthe exoskeleton ofG. lateralis or, skeleton. Concomitantly, the epidermis degrades about
with the exception of44 and 42 kDa protein bands that 75% of the old exoskeleton, the remainder of which is
were in the epicuticle ofC. antennarius, in any layers of shed as an exuvia at ecdysis (reviewed in Skinner, 1985;
theexoskeletonsofthe otherthree species. Duringproec- Stevenson, 1985; Skinner et al. 1991).
dysis, the membranous layeriscompletelydegraded, and The exoskeleton ofan anecdysial (intermolt) decapod
proteins 31 kDa or smaller are preferentially degraded crustacean consists of four layers. From the outside in
from theexocuticleand endocuticle oftheoldexoskeleton theyareepicuticle, exocuticle, endocuticle, and membra-
ofG. lateralis, which is cast as an exuvia at ecdysis. The nouslayer.Theepicuticleisthin [about 7^m;thicknesses
relativeamountsofproteinsinextractsofepicuticle from are given forthe Bermuda land crab Gecarcinus lateralis
(1) anecdysial exoskeletons and (2) exuviae were very (Skinner, 1962)] and composed of proteins, lipids and
similar, suggestingthat there was little degradation ofep- calcium salts, but no chitin (Travis, 1955; Welinder,
icuticle during proecdysis. Some of the proteins of the 1975b;Stevenson, 1985). Theepicuticleisthoughttoren-
three inner layers ofthe exoskeleton ofG. lateralis have der the exoskeleton impermeable (Mary and Krishnan,
characteristicssimilartothoseofflexiblecuticlesofinsects; 1974), preventing both water loss and solubilization of
they have acidic pis and they form "charge trains," i.e.. exoskeletal components in aqueous habitats (Dennell,
1960). The exocuticle (about 30 ftm) isa calcified matrix
ofchitin and protein. The epicuticle and exocuticle form
Received the soft shell of the newly exuviated crab. These layers
' Present address: Department ofBiological Sciences, University of
South Alabama, Mobile, Alabama 36688. are thought to harden during metecdysis by tanning
-Correspondingauthor. (Vaccaand Fingerman, 1975a,b) andcalcification (Travis,
427
428 O'BRIEN ET AL
J. J.
1960). The endocuticle (about 200-400 ^m) isalso a cal- Durliat, 1986), most of the exoskeletal proteins from
M
cified matrix ofchitin and protein. Its synthesis and cal- Crustacea had r smaller than 30 kDa.
cification begin in metecdysis, several days after ecdysis Finally, different concentrations of 20-hydroxyecdy-
(Stage BofDrach, 1939;seeSkinner, 1962),andcontinue sone, selected to mimic hemolymph concentrations
through Stage C3. The membranous layer (about 20-30 throughout the intermolt cycle, have been tested on the
nm) also contains chitin and protein but is not calcified; synthesis ofcrustacean integumentary proteins, both ex-
it is the innermost layer and abuts the epidermis at the oskeletal and cellular. The effects have been monitored
apical cell membrane. The membranous layer is synthe- in vitro in A. leptodactylus (Bielefeld et ai. 1986; Traub
sized during Stage C, (Green and NefF, 1972), and its etai, 1987)andboth in vitro(Paulson and Skinner, 1991)
completion signals the onset of anecdysis, or intermolt and in vivo(StringfellowandSkinner, 1988)in G. lateralis.
(stage C4; Skinner, 1962, 1985). A discrete membranous TMhe most significant effectswereonthesynthesisofsmall
layer has not been described in reviews ofinsect cuticle r proteins.
(Neville, 1975; Filshie, 1982; Willis, 1987). We have been investigating some of the cellular and
Compared totheexoskeletonsorcuticlesofmanyother moleculareventsin the moltingprocess. They occurdur-
arthropods, the greater size and thickness ofthe decapod ing proecdysis. and include synthesis ofmuscle in regen-
crustacean exoskeleton present distinct advantages for eratinglimbsconcomitant withatrophyofmuscle in che-
isolating individual layers ofthe exoskeleton and ofexu- lae(Skinner, 1966; MyklesandSkinner, 1982, 1985),and
viae. These characteristics of the crab exoskeleton also synthesis of the two outer layers of a new exoskeleton
permitthe recoveryofsignificantamountsofprotein from (epicuticle and exocuticle) concomitant with the partial
individual layers. Itis much moredifficultto separate the degradation oftheold exoskeleton. The mechanisms that
individual layersofthecuticlesofmostinsects(Hackman, control such events must be complex. We focus here on
1974). the composition, synthesis, and turnover ofthe exoskel-
Despitetherecoverabilityoftheseparate layersofcrus- eton.
tacean exoskeletons, amino acid compositions have been In this report, we describe proteins present in the four
determined only for entire exoskeletons of the crayfish layers ofbrachyuran exoskeletons, with an emphasis on
Astacusfluviatilis (Welinder, 1974, 1975a; Herzog et ai, C. lateralis, the exoskeleton ofwhich has been analyzed
We
1975); the crabsScylla serrata (Hackman, 1974), Cancer during several different stages ofthe intermolt cycle.
pagurus, and Carcinus maenus; the Norway lobster show electrophoretic patterns of proteins and compare
Nephrops norvegicits; and the shrimp Penaeus duorarum the proteins present in anecdysial, proecdysial, and me-
(Welinder, 1974). Tyrosine, which is a rather rare com- tecdysial exoskeletons and exuviae of G. lateralis. The
ponentofmost proteins, constitutes 3 to 7.5% ofthetotal distribution ofa number ofthe proteins in one or more
amino acids in most analyses. Such a result might be ex- layersoftheexoskeleton (data shown here), aswell asthe
pected iftyrosine is involved in protein cross-linking in patternsoftheirsynthesis(StringfellowandSkinner, 1988;
crustacean exoskeletons (Vacca and Fingerman, 1975a,b). Paulson and Skinner, 1991)anddegradation (O'Brien and
In general, exoskeletal proteins are rich in glycine, which Skinner, 1987, 1988), make them favorable candidates
may play a role in covalent binding ofprotein to chitin forthe investigation ofgenesspecifically activated during
(Herzog et a!., 1975). Acidic amino acids account forap- the molting process.
proximately 20% ofthe total. The exuvia is much more friable than the anecdysial
Intact proteins of some exoskeletons have also been exoskeleton. The cuticular proteins that are degraded
analyzed. Exoskeletons have either been extracted directly during proecdysis may be involved in the calcification
and their proteins displayed on polyacrylamide gels (S. and maintenance ofrigidity, whereasthose that are "pro-
serrata:Hackman, 1974),orpriortoanalyses, fractionated tected" from degradation may be more important in de-
into three parts: epicuticle plus exocuticle, endocuticle, termining the framework and shape ofthe exoskeleton.
and membranous layer (C. pagurus: Welinder, 1975b).
In both of these investigations, the positions of the ex- Materials and Methods
tracted proteins in polyacrylamide gels after electropho- Experimentalanimals andpreparation ojlayers oftheir
resiswerediagrammed,buttheactualgelswerenotshown.
exoskeletons
The amino acidcompositions ofmixturesoftheextracted
proteins have also been determined (Hackman, 1974; G. lateralis specimens obtained from the Bermuda
Welinder, 1975b). Exoskeletal proteinsofthecrayfishAs- Biological Stationweremaintainedatapproximately25 C
tacus leptodactylus have been sized on one dimensional and 12:12 h light:dark cycle. Precocious molts were in-
polyacrylamide gels (Vranckx and Durliat, 1980, 1986). duced in G. lateralisbyautotomy ofmorethan four limbs
With the exception ofthe latter analysis, in which some (SkinnerandGraham, 1970, 1972). Specimensoftherock
of the proteins were as large as 400 kDa (Vranckx and crab Cancer antennariits, the shield-backed kelp crab
PROTEINS OF CRAB EXOSKELETONS 429
Pugettia producta. and the southern shield-backed kelp amortarand pestle,and membranouslayerswereminced.
crab Taliepusnuttalli, werecollected nearSanta Barbara, MSamplesweremexMtracted in 5 Alguanidinethiocyanate,0.4
California. EDTA, 5 Pipes, pH 7, (1:10, w:v), with gentle
Anecdysial animals were placed on ice for 0.5-2 h to shaking at room temperature for 2-5 h. Extracts were
separate integumentary tissues from the membranous centrifuged at 16,000 X gfor 5 min in an Eppendorfmi-
layer(O'Brien elal.. 1986). Celldebriswasremoved from crofuge, and thesupernatantswererecovered. Samplesto
the membranous layer, and the external surface of the be stormedMorelectrophoresed were dialyzed against Buffer
carapace was cleaned with moist Kimwipes. Epicuticles A (10 ammonium bicarbonate, pH 7.8) overnight at
from anecdysial animals were removed from the dorsal 4C. Samples to be iodinated were dialyzed overnight at
carapace with a power rotor(Li'l Crafty, Sears) equipped 4C against 0.4 Al NaCl, 2.5 mM NaN3, 5 mM Pipes,
with a steel pyramidal bit. Both exoskeleton and rotor pH 7. Following iodination by the chloramine T method
were held inside a plastic bag, which collected airborne (McConaheyand Dixon, 1980), thesamplesweredialyzed
particles ofepicuticle as they were scraped from the car- against Buffer A at 4C overnight until radioactivity in
apace. The procedure was repeated to collect exocuticle. the dialysate was reduced to background. Protein was
In G. lateralis, epicuticle (dark purple in the dorsal car- measured by fluorescence (Avruch and Wallach, 1971);
apace), exocuticle (orange), and endocuticle (white), can aliquots were then lyophilized and stored at 60C. Ly-
be distinguished easily on the basis of color. Following ophilized proteinswere solubilized in appropriate buffers
removal ofthe epicuticle and exocuticle, the endocuticle for 1 or 2D gel electrophoresis. Samples for ash-free dry
wascracked andthe membranous layer(ML) peeled away weights were dried to constant weight at 55C and then
from the inner surface ofthe endocuticle; this provided heated at 500C for 4 to 24 h in pre-ashed aluminum
ML C (membranous layer adjacent to epidermal cells; boats.
Fig. 2, lane 5). The innerlayeroftheendocuticlewasalso
scraped to remove any remnants of membranous layer; Gelelectrophoresis
ML
this provided E (adjacent to endocuticle; Fig. 2, A discontinuous gel system was used for ID gels
lanTeh4e).possibility that particular proteins are associated (poLlayeamcmrlyil,am1i9d70e).(SSterpianrgafteilnlgowgealsndweSrkeignrnaedri,en1t9s8o8f)5o-r159%-
awsitfholpliowgsm.eFntreodmrtehgeiosnasmoefatnhiemeaxloss,kperloetteoinnswawserdeetexetrrmaicnteedd (1O8'%BrpioelnyaacnrdylaSmkiidnen,ert,he1l9a8t8t)er;csotnatcakiinnegdg0e-ls10w%ergelyc4e.r5o%l.
from the pooled three outer layers (epicuticle plus exo- acrylamide. In 2D analyses, proteins were separated in
cuticle plus endocuticle) ofthe highly pigmented dorsal
craegriaopnasceosfotfhaenbercadnycshiiaolstcergaibtse,sa.nTdhferobrmatnhcehiuonstpeiggimteesntaerde gatehclersyfli(ra0smt.i8ddiemm,emn0s.iX2o%n1bb1iys,cimsa)onedlceco5tmr%ipcoafsmoepcdhuosoilnfign9e(IsMEF()Ou'urFseaair,nrgel3tl.u8be%el
lateral extensions ofthe thoracic exoskeleton, and cover al, 1977). Theampholineswerea 1:1:1 blendofpH = 3-
cthoempgirlilscehaamnbeirnsn;erthaenidr uanndeorultyeirngshienetteogfumeepnitdaerrymitsissseupe-s g5,ra4d-i6e,ntanodfp3-H105-(7B.iPor-oRtaedi)nsthwaetrperosedpuacreadteadnianptphreoxsiemcaotned
acneerlral,ts,ed1t9eb6gy2u)m.aelnPatryaoetlreiognlfascnodwsne,nreeacntadilvsheoetemixsotslrueaycmctpoendhtafsiirnnoiumnsgestsh(teSokrtiawngo-e dbwleiurmeee.nsTstoiaoicnnoebdmypwasiirtzheesotihnlevSeerDff(SiWcsralcaayybogefetltsahle.(,Ot'1wF9aor8rs1et)lalio,nrs,1C9os7oo5m)m.aesGsgeeilless
newlysynthesizedouterlayers(epicuticleplusexocuticle) were stained first with Coomassie blue, destained, and
of unpigmented regions ofbranchiostegites, moderately restained with silver. The relative intensities ofbands in
pigmented lateral branchiostegites, and the purple dorsal stained ID gels were quantified with an LKB 2202 UL-
rfeegwihonouorfstfhoellcoawrianpgaeccedsysoifsmdeutreicndgysSitaalgeaAni(mDarlaschw,ith1i93n9;a TcoRmOpuStCeArN(sclaanssernodtensshiotwonm)e.teArutaotrtaadcihoegdratophaynwaAsppalsedeI-I
Skinner, 1962). Exuviae, remnantsoftheoldexoskeleton scribed (Stringfellow and Skinner, 1988; Paulson and
from which the animal emerges at ecdysis, are lined by a
Skinner, 1991).
transparent ecdysial membrane (Stevenson, 1985) which
was removed. Epicuticles, exocuticles, and endocuticles
Defection ofcalcium-bindingproteins
were recovered from exuviae by careful chipping with a
sharp scalpel. During vacuum desiccation, pieces ofepi- Calcium-binding by proteins in gels was detected ac-
cuticle separated spontaneously andcleanly from exuviae. cordingtothemethodofMaruyamaetal. (1984). Proteins
(75-100 jig/lane) extracted from specific layers ofG. la-
Extraction ofproteins teralis exoskeleton were separated by SDS-PAGE. Gels
Exoskeletal layerswere dried at room temperature. The weremeqMuilibrated in transferbuffer(25 mMTris, pH 8.5,
three outer calcined layers were pulverized separately in 192 glycine, 20% methanol) with 3 (100 ml) X 20
430 J. J. O'BRIEN ET AL.
min washes. Proteins were transferred to 0.1 nm (pore Table I
size) nitrocellulose membranes (Schleicher and Schuell) CharacteristicsoflayersofexoskeletonofGecarcinuslateralis
with aTrans-Blot Electrophoretic TransferCell (Bio-Rad)
4C
at for 16 h at 60 V. Membranes were washed with
10mMimidazole-HCl,pH 6.8,60mAlKC1, 5 mA/MgCl,
(Buffer B), 3 (100 ml) X 20 min then soaked 10 min in
50 ml Buffer B with 1 mCi/L 45Ca++ (New England Nu-
clear), washed for 5 min in 500 ml 50% ethanol anddried
at room temperature.
Results and Discussion
The intermolt cycle ofa decapod crustacean isdivided
into an intermolt period (anecdysis), a premolt period
(proecdysis), ecdysis, and a postmolt period (metecdysis).
Two layers ofthe new exoskeleton, the epicuticle (out-
ermost layer)and theexocuticle (the next innerlayer)are
synthesizedduringproecdysis. Theanimal emergesatec-
dysis clad in these two layers only; the partially degraded
old exoskeleton is shed as an exuvia. In metecdysis, syn-
thesis ofan endocuticle begins. In G. lateralis, formation
oftheendocuticlecontinuesfora month orsoafterwhich
synthesis ofthe innermost layer, the membranous layer,
begins. The completion ofthe membranous layer signals
the beginning ofanecdysis.
Characteristics ofthe layers ofthe anecdysial (intermolt)
exoskeleton
Somecharacteristicsofthefourlayersoftheanecdysial
exoskeleton ofG. lateralisare listed in Table I. Epicuticle
constituted about 15%, exocuticle 8%, endocuticle 75%,
and membranous layer only about 2% ofthe dry weight.
The large contribution ofthe epicuticle may be related to
its role in inhibiting water loss. Desiccation is a major
influence affecting adaptation ofcrabs to a terrestrial en-
vironment (Wolcott, 1988)and G. lateralishasoneofthe
lowest ratesofevaporative waterlossamongbrachyurans
(Greenaway, 1988). Because epicuticle isonly onequarter
the thickness ofexocuticle but has twice the dry weight,
the water content of the native epicuticle must also be
very low.
Theconcentration ofextractable protein perunit mass
ofdry weight was lowest in the epicuticle and increased
progressively with each deeperlayer. Although the inner-
most membranous layer contributed the least to the rel-
ative weight ofthe exoskeleton, perhaps due to the lack
ofcalcium salts, it yielded the most extractable protein
per unit mass. Ash-free dry weights of the exoskeleton
measure the relative composition of inorganic material,
chiefly calcium salts in the form ofcalcite (Roerand Dil-
laman, 1984). Theepicuticleand exocuticle ofG. lateralis
had slightly higher levels of inorganic material than the
underlyingendocuticle; the relativeash-free composition
PROTEINS OF CRAB EXOSK.ELETONS 431
X X N ML ML
kDa kDa kDa
:
....
31
31,
28 ^28- I
28-
25 25- 25-
23 23'
16 16- -16-
.14 14 15-
13 14-
11-
10
6 7 8
Figure 1. Comparison ofGecarcinus lateralis exoskeletal proteins stained with eitherCoomassie blue
orsilver.Lanesarefroma 1Dgel(9-18%acrylamide;0-10%glycerol)onwhichwaselectrophoresedproteins
(30^g/lane) ofeach ofthe four exoskeletal layers ofan anecdysial crab. Afterelectrophoresis, thegel was
stained with Coomassie blue, photographed, destained, then stained with silver and photographed again.
The lanesfrom the two photographswerecut outand thetwodifferently stained lanesofeach ofthe four
exoskeletal layers were placed adjacent to each other. Lane 1, low molecular weight standards (Bio-Rad),
stainedwithCoomassieblue. Lanes2-9,layersfromexoskeleton;lanes2,3,epicuticle,P;lanes4,5,exocuticle,
X; lanes 6, 7, endocuticle, N; lanes 8, 9, membranous layer, ML. Lanes 1, 2, 4, 6, and 8, stained with
Coomassie blue; alternate lanes stained with silver. Approximate sizes of major protein bands equal or
smaller than 55 kDa are indicated between the paired lanes; a dash indicates in which lane the band is
present.Proteinslargerthan55kDacouldnotbeaccuratelysizedbecauseahighconcentrationofacrylamide
M
hadbeen usedand only low rstandards had been run.
shown in Fig. 4) was loaded on each IEF tube gel and Although radioiodination isthe most sensitive method,
electrophoresed, after which the second dimension gels theintensity ofthebandsobserveddependsontheamino
(9-18% acrylamide) were stained with Coomassie blue. acidcomposition oftheproteins. Tyrosines, andtoalesser
In the gel ofthe epicuticle, only a group offive proteins, extent histidines, are the primary targets of iodination
ranging in size from 54 to 42 kDa, referred to here asthe and proteinsrich in theseaminoacidsarethe most readily
epicuticularquintet, could be detected (data not shown). visualized. In addition, iodination may cause structural
No bands were seen in the gels ofthe other layers even modifications that may altertheelectrophoretic mobilities
after attempts to maximally enhance visualization ofthe ongelsand,consequently, thebandingpatternsofproteins
proteins. in comparison to the banding patterns ofuntreated pro-
In preparation foraminoacid sequencingofindividual teins that are electrophoresed and then visualized by
protein spots, large amounts ofprotein (250 jig/gel), iso- stainingwitheithersilverorCoomassieblue. Nevertheless,
lated from each ofthe four exoskeletal layers were elec- when populations ofproteinsare similarly treated, iodin-
trophoresed on a second set of 2D gels. The gels were atedinthiscase,thesamestructural modificationsshould
blotted on PVDF(polyvinylidene difluoride) membranes, be sustained by the same proteins in each population.
which have a higher binding capacity than othertypes of Thus, meaningful comparisons can be made among
membranes (Graddis, 1990). Fixation with 0.5% glutar- preparations analyzed by the same method.
aldehyde (Karey and Sirbasku, 1989) further increased
the retention of the proteins to the filters. When such AenxaolsykseliestoofneaxntrdacetxaubvlieapbroyteIiDnsgeilnealneecctdryospihaolresis
blotswere stained with Coomassie blue, manyspotswere
seen (Table III) that were not detected when smaller In the following, proteins ofanecdysial (intermolt) an-
quantities ofprotein were analyzed. imals are considered first, then proteins of proecdysial
432 J. J. O'BRIEN ET AL.
(priorto ecdysis) and metecdysial (afterecdysis) animals.
In each category, layers ofthe exoskeleton are described
kDa
from most external to most internal, i.e., epicuticle, ex-
ocuticle, endocuticle, and membranous layer.
The members ofthe epicuticular quintet were promi-
nentinbothanecdysialepicuticleandepicuticleofexuviae
(Fig. 2, lanes 1, 6, 7). Exuviaecontain epicuticleand rem-
nantsofexocuticleandendocuticle, but littleifanymem- 54,
branous layer, most or all of which is degraded during 50
proecdysis. The presence ofthe epicuticular quintet was 4448
correlated with the extent of pigmentation (see Fig. 3).
Other proteins ranging from 94 to 80 kDa that were
prominent in anecdysial epicuticle, exocuticle, and en-
docuticle were missing from epicuticle of exuviae and 25
markedly reduced in the other two layers (Fig. 2). Other
than lacking the 94-80 kDa proteins, the profiles ofepi-
cuticle protein extractswere very similar before and after
ecdysis.
Extractsfromexocuticle,endocuticle,and membranous
layerfrom anecdysial G. lateraliscontained many protein
ML
PXNECPPXN
kDa kDa skeFliegtuonr.eP3r.oteEipniscuetxitcrualcatredqufirnotmetPo+ccXurs+iNn poifgtmheenbtreadncrheigoisotnesgiotfeeaxnod-
S 177 dorsal carapace regions ofGecamnus lateralis. SDS silver-stained gel
' -164 (9-18%acrylamide,0-10%glycerol); 7.5 ngprotein/lane.C.dorsalcar-
-138 apace;B,branchiostegite. NumberstoleftindicateM,s(kDa)ofprotein
. -118 bands. Lane 1,anecdysialpurpledorsalcarapace;lane2.anecdysial un-
pigmented hranchiostegite; lane 3. metecdysial purpledorsal carapace;
-
80 lane4. metecdysial unpigmented branchiostegite.
- 68
-
54 55 bands 31 kDa or smaller (Fig. 2, lanes 2-5). Although
50r I II protein bands ofthese sizes formed only traces in epicu-
48V
44T ticle, many were prominent in the other three layers.
42' Whilethisresultpoints upadifferent composition forthe
M
31' epicuticle, the correspondence of rs ofproteins in the
28
25- otherthree layersdoes not necessarily imply theiridentity.
23 Protein bands of 16-10 kDa were very conspicuous in
16 both fractions ofthe membranous layer (ML E, closer to
14 ML M
endocuticle, and C, closertoepidermal cells); low r
23456789 bands were also conspicuous in exocuticle and endocu-
M
ticle. Most ofthese major low r protein bands as well
ANEC. EXUVIA as a prominent 25 kDa band in exocuticle, endocuticle,
and MLC, were presentonlyintraceamountsormissing
ofFtihgeureexu2v.iaPorfote(jicnasiiirnnuain.e\cdlyusiii-arlulil*a.yeSrislovfert-hsetaeixnoesdkeSlDetSongealnd(5l-a1ye5r%s in extracts from exuviae (Fig. 2, lanes 6-9). AMs a conse-
acrylamide),7.5/jgprotein/lane.AbbreviationsasinFigure 1.Numbers quenceofthepreferentialdegradationofsmall rproteins
toleftand right oflanesindicate M,s(kDa)ofprotein bands. Lanes 1- in vivo during proecdysis, a number ofother proteins 55
5. layers from exoskeleton ofanecdysial animal. Lane 1, P; lane 2, X; kDa or larger were relatively more prominent in extracts
lane 3, N; lane 4, membranous layer adjacent to endocuticle, ML E; from exocuticleandendocuticleofexuviaethan from the
ltiasnseue5s,,mMeLmbCr.aLnaonuess6la-y9e.rlaadyjearscefnrtotmoeexpuivdiae.rmLaalnceel6l,seopficiunttieclgeumiesnoltaatreyd same layers from anecdysial animals.
byvacuumdesiccation, P; lane7,epicuticleisolated bychippingwitha Protein patterns in exocuticle extracts were highly re-
scalpel. P; lane8, X; lane9, N. producible. The gel shown in Figure 1 was 9-18% acryl-
PROTEINS OF CRAB EXOSKELETONS 433
amide and 0-10% in glycerol; the gel in Figure 2 was 5- and electrophoresed on polyacrylamidegels(Stringfellow
15% acrylamide. The different concentrations of acryl- and Skinner, 1988). Both proteins are labeled with both
amide in thetwogelsfacilitatethecleardisplay ofproteins leucineand methionine at relatively high ratesthroughout
M M
oflow r in Figure 1 and those ofhigh r in Figure 2. the intermolt cycle. During the stagesofproecdysiswhen
Comparison ofthe silver-stained lanes ofall four layers epicuticle and exocuticle are synthesized (Di.eariy and
iMnLcluEdianngdtMheLmCemwebrreannootusseplaaryaetreod)fwFiitghurtehe1s(aimnewlhaincehs aDbl2,yi,ate)t.haitncoofrploeruactiinoenmoofremetthhainondionuebliensc,reaansdesthcaotnsoifdetry--
in Figure 2 (membranous layeroperationally subdivided rosine, undetectable during anecdysis and early proec-
ML ML
into E plus C) indicates the presence of many dysis,isalmostashighasthatoftheothertworadiolabeled
M
bands ofsimilar r. As mentioned above, many ofthe aminoacids(StringfellowandSkinner, 1988).Theabsence
protein bands that stained with silver did not stain with ofsignificant stainable quantities ofthe 54 and 42 kDa
Coomassie blue; in particular, note those larger than 16 proteins from all layers ofthe exoskeleton ofG. lateralis
kDa seen only as traces or not seen at all in exocuticle other than epicuticle may be due to a rapid turnover of
and endocuticle, and those larger than 28 kDa also seen theseproteinsascompared to longer-lived structural pro-
only as tracesor not seen at all in the membranous layer. teins ofthe exoskeleton. Alternatively, these may not be
Evidence that the protein extractions are reproducible is structural proteins ofthe exoskeletal matrix but may be
also seen in Figure 2, lanes 6 and 7; arrays ofproteins on metabolically active proteinsin epidermal cellsofthe un-
IDgelsofextracts ofepicuticle from G. lalera/isthat had derlying integumentary tissues or in extensions of cells
been removedfrom exuviaebyeitherascalpel orvacuum within pore canals that permeate the exoskeleton (Green
desiccation wereindistinguishable from oneanother. Ep- and Neff, 1972; Halcrow, 1976; Roer, 1980).
icuticle from highly pigmented regionsofanecdysial exo- In additiontothequintet, amajor25 kDa protein band
skeletons (Fig. 1, lanes 2 and 3; Fig. 2, lane 1; Fig. 3, lane is seen in samples of pooled epicuticle plus exocuticle
1), exuviae (Fig. 2, lanes 6 and 7), and metecdysial exo- plus endocuticle from both pigmented and unpigmented
skeleton (Fig. 3, lane 3)ofG. lateralis, thelatterextracted regions ofanecdysial crabs (Fig. 3, lanes 1 and 2). The
one day after ecdysis, contained the proteins ofthe epi- sources ofthe 25 kDa protein band are, in all likelihood,
cuticular quintet. exocuticle and endocuticle; note the intense staining of
As previously described (O'Brien and Skinner, 1987, the 25 kDa protein in those two layers relative to that in
1988), we have isolated from integumentary tissues two epicuticle (Fig. 2, compare lane 1 with lanes 2 and 3).
alkaline cysteine proteinase activities (ACPs) and two The protein is not seen immediately after ecdysis (stage
proteinaseswithacidicpH optima(APs)thatpreferentially A ofmetecdysis) even though the newly synthesized ex-
degraded membranous layer proteins 26 kDa or smaller. ocuticle is present. Since the protein is extractable from
M
The absence ofsmall r proteins from exuviae (Fig. 2, anecdysial exocuticle, which should be considerably more
lanes 8 and 9) suggests thatsome (orall) ofthe ACPsand rigid than metecdysial exocuticle, it seems unlikely that
APs isolated from integumentary tissues participate in the the protein is not extractable because the exocuticle has
degradation of the exoskeleton of the land crab during begun to harden (Fig. 3, lanes 3 and 4).
proecdysis. Other proteinases, as yet unidentified, may
also contribute to the degradation. Distinguishing individualproteins by2D gel
electrophoresis: anecdysialexoskeleton
Correlation ofthe epiculicular quintet withpigmentation
Analysis of2D gels ofradioiodinated (Fig. 4; Table II)
Theepicuticularquintet, which wasnot seen in extracts exoskeletal proteins fromeach layergaveasomewhatdif-
ofanyotherlayer, wasprominent inextractsofpigmented ferent pattern ofoverlaps than the analysis of2D gels of
dorsal carapace of both anecdysial (epicuticle plus exo- proteins stained with Coomassie blue (Table III). Nev-
cuticleplusendocuticlecombined; Fig. 3)and metecdysial ertheless, the principal conclusion is the same in either
animals (epicuticle plus exocuticle combined), but much case: proteins31 kDaorsmallershowextensivesimilarity
M
less prominent in extracts of unpigmented regions of (percentage ofproteins ofequivalent r and pi) among
branchiostegites ofanecdysial and metecdysial animals. the layers, while proteins larger than 31 kDa show very
A 48 kDa protein band conspicuous in unpigmented re- muchlesssimilarity. Inanycase,whethertheoverlapping
gions ofbranchiostegites from metecdysial animals (Fig. proteinsarein factidenticalawaitsaminoacidsequencing.
3, lane 4) is discussed in the section on proteins from Epicuticularproteins(Fig. 4A),especiallythose31 kDa
metecdysial animals, below. orsmaller, displayedthemostdistinctpattern whencom-
Two proteinsof54 and 42 kDaare majorcomponents pared to proteins extracted from the other three layers.
in extracts ofintegumentary tissues maintained in vitro, Epicuticle differs from the other three layers in: (1) the
labeled with 'H-tyrosine, 3H-leucine, or 35S-methionine presenceoftheepicuticularquintet(Figs. 1-3);(2)having
434 J. J. O'BRIEN ET AL.
138 B
118
88
55
,
28
24
16 . 16
16
14
14
10 11
10
138
118
88 88
68
55 55
28 28 28
25
24
23
16
*
16
16
16
14
14 14
11
10
11
10
Figure4. Analyseson 2Dgelsofproteinsfrom thefourlayersoftheanecdysialexoskeletonofGecaranus
laterahs. Autoradiographs of2D gels. SDS gel for second dimension was 9-18% polyacrylamide, 0-10%
glycerol. Approximate M,s (kDa) ofproteinsare indicated adjacent tospots. (A) epicuticle; 3 X 106cpm,
0.81 jig proteins. Streak in upper right corner isan artifact. (B) As(A)except 15.2 ^gexocuticle proteins.
(C) As (A) except 10.6 ^g endocuticle proteins. (D) As (A) except 13.2 ng membranous layer proteins.
AutoradiographsA-Cexposed 48 h; Dexposed42 h.
PROTEINS OF CRAB EXOSKELETONS 435
Table II
Overlapon2Dgels(similarM,amipi) ofproteins maimedfromIliefourdifferent layersofanecdysialGecarcinuslateralise.\oske/elon
andlabeledwith i:il
436 J J. O'BRIEN ET AL
teins ofapproximately 90 kDa (PCP-28) that are specific i:5I-labeled protein in the 14 kDa charge train in the ex-
to pupal cuticles of M. sexta were also labeled by 3H- ocuticles and endocuticles focused in the acidic region of
leucine and focused atabout the same pi (Kielyand Rid- the gel, whereas the most heavily labeled 14 kDa protein
diford, 1985)asdidthecrabprotein. The 88 kDaproteins in membranous layer focused in the neutral to basic re-
were not degraded in vivo or in vitro by proteinases en- gion. It appearsthat proteins from the membranouslayer
dogenousto the membranous layer(see Fig. 5 in O'Brien are comprised more ofneutral rather than acidic amino
and Skinner, 1987) and were prominent in iodinated ex- acids. These data agree with those ofWelinder (1975b),
tracts ofexuviae ofG. lateralis (data not shown). who reported fewer acidic amino acids in proteins ex-
For Coomassie blue stained proteins smaller than 31 tracted from the membranous layer than from epicuticle
kDa (Table III), the degree ofsimilarity was 36 to 68% plus exocuticle or endocuticle. Cox and Willis (1987a)
among extracts from the exocuticle. endocuticle, and described threegroups ofproteins from flexible insectcu-
membranous layer. Twenty-three of the 49 proteins in ticle: (1)thosewith acidicpis, (2)thosethat formedcharge
exocuticle matched 23 ofthe 45 proteins in endocuticle. trains,and(3)thosethat formedelongated vertical streaks.
The proteins(total)inextractsofmembranouslayerwere Crab exoskeletal proteins showed similarities to the first
also quite similar in size and pi to those extracted from two groups ofinsect proteins, but not to the third. Such
exocuticle and endocuticle. Ofa total of45 membranous streaks are observed in gels that contain urea in the re-
layer proteins, 41% were similar to proteins ofexocuticle solving gel, which ours did not.
and 44% to those ofendocuticle. Welinder (1975b) categorized the exoskeletons of ar-
thropodsas"soft" or"hardened"; softcuticles hada high
Charge trains inproteins ofanecdysial exoskeletal chitin content (63-72%) and proteins that were ( 1) rich
in acidic amino acids, (2) poorin non-polaramino acids,
layers
and (3) highly soluble. The first characteristic isone found
Proteins that are approximately the same size, but that by Willis( 1987)tobetrueofproteinsof"flexible" cuticle
differ in net charge, form charge trains in isoelectric fo- ofinsects. By contrast, hardened crustacean exoskeleton
cusing gels. All four layers ofthe exoskeleton from anec- had a low chitin content and proteins that were (1) rich
dysial crabs had proteins with acidic pis, and some ofthe in nonpolar amino acids and (2) less soluble (Welinder,
proteins from the three internal layers formed charge 1975b). Welinder observed that the exoskeletons of all
trains between pH 5 and 7 (Fig. 4). These patterns are crustaceans he examined were "soft," even though they
similar to those ofproteins extracted from flexible insect are physically hard due tocalcification. Ourdata indicate
cuticle (Andersen el al, 1986; Cox and Willis, 1987a,b). that manycrustaceanexoskeletal proteinsareacidic, sim-
There were more charge trains in membranous layer ex- ilar to those of flexible insect cuticle; thus they support
tractsthan in extracts from otherexoskeletal layers; these Welinder's observation that crustacean exoskeleton has
were at 28, 25, 24, 16, 14, 11, and 10 kDa (Fig. 4D). A some ofthe characteristics of"soft" cuticle.
28 kDa protein band, minor in both exocuticle and en-
docuticle (Figs. 1, 2), focused in the acidic region ofthe Proteins ofnewly synthesized exoskeleton: metecdysial
gels(Fig. 4B,C). Themajorcomponentofa28 kDacharge
train in the membranous layer focused in the neutral to Proteinswereextracted from thenewlysynthesizedep-
basic region (Fig. 4D), similar to a 27 kDa charge train icuticle plus exocuticle which encase the animal as it
in the innermost layersofM. sexlaendocuticle (Wolfgang emerges at ecdysis. Those proteins that became heavily
and Riddiford, 1986), equivalent tothe crustacean mem- iodinated had acidic pis; all except members ofthe epi-
branous layer. In ID gels stained with silver, the 25 kDa cuticular quintet migrated to the bottom ofthe IEF tube
protein band was very intense; the 24 kDa band barely gel (Fig. 5). Although threeofthe I25l-labeled metecdysial
detectable. The 24 kDa proteins may thus be expected to proteins(Fig. 5; 57, 54,and48 kDa)hadsimilarmobilities
be very rich in tyrosine or histidine and consequently be toproteinspresent in extractsofanecdysial epicuticle(Fig.
more highly labeled in vitro with I25I. In fact, in integu- 4A), none was similar to those in anecdysial exocuticle
mentary tissues removed from animals at the time that (compareFig. 5with Fig. 4B). Perhapstheproteinsmissing
eapnidcuDti:cJlalec)aannddienxoccuubtaitceldewairteh b'eH-itnygrossyinnteh,easi2z4edkD(aD2p,eraori-y afnrdomtheexrtreabcytsreonfdetrheedaniencsdoylusbilael ilnaygeurasnhiadivneebteheinoctyaannnaetde
teinbandisveryheavilylabeled(StringfellowandSkinner, as appears to have happened to a 48 kDa protein band.
1988). The 24 kDa charge train was specific to the exo- It was prominent in extracts from comparable regions of
cuticle and membranous layer but was present in only the exoskeleton, i.e., unpigmented branchiostegites of
trace amounts in the former (Fig. 4B, D). metecdysial animals (Fig. 3, lane 4), but wasabsent from
Proteinsextracted from the innerthreeexoskeletal lay- branchiostegites of anecdysial animals (Fig. 3, lane 2),
ersalso had 16and 14kDachargetrains. The mostheavily suggesting that it had been altered during tanning ofthe