Table Of ContentReference: Biol. Bull 182:409-415. (June, 1992)
Proline Synthesis During Osmotic Stress in Megalopa
Stage Larvae of the Blue Crab, Callinectes sapidus
RONALD BURTON
S.
Program in Evolutionary Biology. Department ofBiology. University ofHouston,
Houston, Texas 77204-5513
M
Abstract. The freeaminoacid(FAA)pool ofindividual inexcessof0.1 in seawater-acclimated Crustaceawhere
Callinectes sapidus megalopas acclimated to 100% sea- the total FAA pool may account for as much as 50% of
wateraveraged over56% largerthan that of50% seawater thetotal intracellular osmolyte pool (Bowlusand Somero,
acclimated megalopas. Most ofthe difference was due to 1979).
a four-fold increase in prolineconcentration at the higher While changes in FAA pool sizes have been widely
salinity. In 100%- seawater, proline comprises 64% ofthe documented, relativelylittleisknownabouttheregulation
total FAA pool in megalopas; this contrasts with the role ofFAA concentrations during osmotic stress. The most
ofproline in adult tissues where it never exceeds 25% of widely cited hypothesis involves the direct action of in-
the total FAA pool. Metabolic tracer studies using 14C- organic ions on a key enzyme, glutamate dehydrogenase
glucose and l4C-glutamate as radiolabelled precursors (GDH, EC 1.4.1.2), which catalyzes the reductive ami-
showed that dc novo synthesis of proline was very low nation of -ketoglutarate to form glutamate (see Gilles,
unless induced by hyperosmotic stress. The induction of 1979; Gillesand Pequeux, 1983; Hochachka and Somero,
the synthetic pathway was inhibited by cycloheximide, a 1984). Increasing medium salinity is postulated to result
protein synthesis inhibitor. These results suggest that the in increases in intracellular NaCl concentrations that may
GDH
induction ofproline synthesis is regulated by the synthesis directlystimulate activity, resultinginthesynthesis
ofeither one ofthe enzymes catalyzing the three steps in ofglutamate. Becauseglutamate istheaminogroupdonor
the glutamate to proline pathway or a protein acting to for synthesis ofalanine and aspartate (and probably gly-
stimulate the activity ofone ofthose enzymes. cine)and adirect precursorforproline, the increased glu-
tamatesynthesiscould drive, by massaction, the synthesis
Introduction oftheseotherFAAs. OtherkeyenzymesinFAAsynthesis
are unaffected by changes in inorganic ions (e.g., trans-
The adjustment ofintracellular free amino acid (FAA) aminases), while some involved in FAA catabolism are
concentrations plays an important role in acclimation to inhibited by increased inorganic ion concentrations(e.g.,
salinity change in Crustacea and a diversity ofother ma- serine hydrolyase). Combined, these effects are thought
rine invertebrate taxa (see reviews by Florkin and Schof- to alterthe synthesis/catabolism balance for FAA and re-
feniels, 1969;Gilles. 1975, 1979; Schoffeniels, 1976). High sult in their accumulation.
intracellular FAA concentrations apparently function to Unfortunately, while data continue to support the oc-
balance high inorganic ion concentrations in the hemo- currence ofde novo synthesis ofFAA in response to hy-
lymph ofanimalsexposed toelevated salinity. Only afew perosmotic stress (e.g.. Burton, 1986), few data directly
non-essential amino acids such as alanine, proline, and support theabove model forregulation ofFAA synthesis.
glycine are major contributors to the response and show Theeffect ofNaCl and otherinorganic ionson glutamate
rapid, quantitatively important, changes in concentration dehydrogenase activity in vitro has proven tobe complex
followingchanges in environmental salinity. One or more atbest,and some investigators nowproposethatthe major
oftheseFAAtypicallyobtain intracellularconcentrations change leading to FAA accumulation is not increased
synthesis, but rather reduced FAA catabolism (Gilles,
Received 19 November 1991: accepted 10 March 1992. 1979;Gillesand Pequeux, 1983). However, Burton ( 1986,
409
410 R. S. BURTON
199la, b) has shown that in the euryhaline intertidal co- While adult C. sapidus is too large for the in vivo radi-
pepod Tigriopuscalifornicus, detectable proline synthesis otracer studies needed to address mechanisms ofproline
isobserved onlyduringhyperosmoticstress. Afterproline synthesis, C. sapidus megalopas (dry weight ofapproxi-
accumulates for approximately 24 h, proline synthesis is mately0.4 mg)arelocallyabundantandeasilymaintained
effectively turned off. This is in contrast to other FAAs in the lab. In the work described below, osmotically in-
(such as alanine, glutamate, and aspartate), which are duced changes in FAA concentrations are documented
synthesized continuously under a variety of salinity re- in C. sapidus megalopas and the role ofprotein synthesis
gimes. Clearly, proline synthesis is not simply driven by in regulating these changes is assessed via in vivo appli-
mass action following increased glutamate production cation ofthe protein synthesis inhibitor cycloheximide.
because glutamate production occurs under all salinity
conditions and glutamate pool size does not change Materials andMethods
markedlyduringhyperosmoticstress. Similarly, regulation C. sapidusmegalopaswerecollected withahand-pulled
of the proline pool cannot be the result of changes in beam trawl in shallowwater(<1.5 meter)alongthesandy
prolinecatabolism alone, becausesuchamodelcould not Gulfcoastbeach ofGalveston Island, Texas, inearlyJune
account forthe fact that 14C-labelling ofglutamate (from to August 1991. Ambient salinities ranged from 17 to 37
labelled bicarbonate) occursunderconstant salinity (50% ppt. Animals were maintained at room temperature
or 100% SW), while no labelling of proline is observed (23C) and acclimated for 3-5 days at 17 ppt (50% sea-
under these conditions (Burton, 1986). These data are in water = 50% SW) and 34 ppt (100% SW) before being
direct conflict with the mass action synthesis model dis- exposed to experimental treatments. Animals were fed
cussed above. commercial flake fish food(Tetramin)duringacclimation.
Recently, by using in vivotranslation inhibitorstudies, C. sapidus megalopas were initially identified by com-
we have shown that the induction ofproline synthesis in paring them to the description presented in Costlow and
T. californicusin responsetohyperosmoticstressrequires Bookout(1959). Numerous megalopas moltedtothefirst
protein synthesis (Burton, 199Ib). By providing I4C-(U)- crab stage in our aquaria within a few days of capture;
L-glutamate asa proline precursor, evidencewas obtained thesewereidentifiedasC. sapidusasdescribedin Williams
that the ultimate site ofaction for protein synthesis in- (1984).
hibitorswasin thethree-step pathway between glutamate Procedures for studying the incorporation of labelled
and proline. This work suggests that hyperosmotic stress precursors intothe FAA pool in individualsofC. sapidus
induces the synthesis ofone or more ofthe enzymes in were as follows: prior to exposure to precursor-laced me-
theglutamate to proline biosynthetic pathway ora protein dium, animals were pretreated for 1 h with an antibiotic
that stimulates the activity ofthese enzymes. Given that mixture ("AM 4" ofProvasoli el ai, 1959) in filtered (0.2
mM
this mechanism for the regulation of FAA metabolism A/), buffered (30 HEPES) commercial (Instant Ocean)
has not been previously documented among marine artificial seawater(SW) ofappropriate salinity. The effec-
Crustacea, it was of interest to determine the generality tiveness ofthis antibiotic mixture in preventing contam-
ofour T. californicuswork by performing similar studies inating bacterial growth was previously tested (Burton,
on other, taxonomically distant, crustacean species. 1991a). Radioactive precursor, l4C-(U)-L-glutamate,
Several criteria were important in choosing an appro- (Sigma Chemical Company, 229.4 mCi/mmol) or I4C-
priatestudysystem fortestingthemechanism ofinduction (U)-D-glucose, (Sigma Chemical Company, 255 mCi/
ofproline synthesis. First, the test organism should be a mmol) was added to a small volume (5 yuCi/150 ^1) of
euryhaline osmoconformer where adjustment of FAA medium ofappropriate salinity; all media contained the
concentrations function in salinity acclimation. Second, antibiotic mixture. Experimental treatments involvingthe
because proline is only a minor constituent ofthe FAA translation inhibitor cycloheximide also used a 1-h pre-
poolinsomespecies,aspecieswasneeded inwhichproline treatment period (with antibiotics and cycloheximide)
was known to be an important contributor to the FAA priorto salinity transfer. Transfers between pretreatment
pool. Finally, foranalyticconvenience, we soughtasmall and treatment media were carried out by pipetting indi-
organism because smallerquantitiesoftracerisotopesare vidual megalopas ontofilterpaperandthen movingthem
necessary formetabolic studies. Onesystem meetingthese (with a fine forceps) into 1.5 ml microcentrifuge tubes
criteria isthebluecrab, Callinectessapidus. an abundant containing the desired treatment medium. Up to six me-
portunid thatexperiencessubstantial salinity variation in galopas were treated together in a single tube. Handling
its natural estuanne habitat along the Texas coast. The was identical for controls and treatments and did not di-
participation ofFAA in osmotic acclimation ofadult C. rectly result in any mortality. Osmotic concentrations of
sapidus has previously been studied (Gerard and Gilles, artificial SW solutions were routinely determined with a
1972; Engel, 1977), and proline was found to be a major hand refractometer and checked with a vapor pressure
contributor to the osmolyte pool in each tissue studied. osmometer (Wescor Model 5500).
REGULATION OF PROLINE SYNTHESIS 411
Following experimental exposures (typically 3-6 h), Results
animals were individually sacrificed and FAA extracted
Sinam1p0l0esn\woefre80r%eseutshpaennodledanidn t6h0enn\dorfie0d.1unMdesrovdaicuumumbi.- FtoA5A0p%ooalndo/1Ca0!0l%ineScWtes sapidus megalopas acclimated
carbonate and then reacted with 40 n\ ofdansyl chloride Following collection from ambient 100% SW, groups
in acetone (0.5 mg/ml) for 90 min at room temperature ofmegalopas were acclimated to 100%. and 50% SW for
to fluorescently label primary and secondary amino fivedays asdescribed above. Results ofFAA analyses are
groups. FAAanalysiswascarried outondansylderivatives shown in Figure 1. In 50% SW, taurine and glycine are
ofthe FAA usingreverse-phase high pressure liquid chmrom- the dominant FAAs, comprising approximately 42% and
matography (HPLC) (CIS "Hypersil" 5 /u 4.6 X 250 25% ofthe measured pool, respectively. The total FAA
cartridge column. Alltech Assoc.) with fluorescence de- pool of 100% SW acclimated animals averaged over 56%
tection; peaks representing FAA were quantified with a largerthan thatof50%< SWacclimated animals(one-tailed
computing integrator and were individually collected di- /-test, P = 0.022). The only amino acid that contributed
rectly into minivials for liquid scintillation counting (see significantly to the increased pool was proline (taurine
HPLC
Burton, 1986, forfurther details). Although glycine, and glutamate actually showed relatively minor but sta-
taurine, alanine, and proline derivatives were completely tisticallysignificantdecreases);in 100% SW, prolinecom-
resolved, there was some difficulty in resolving dansyl- prised over 64% ofthe FAA pool.
glutamate from dansyl-aspartate; data for the combined
glutamate/aspartate peak are presented here as "gluta- Incorporation ofradiolabelledglucose into the FAA pool
mate." To determine whether the confounding ofgluta-
mate and aspartate would have a significant effect on es- Megalopas were presented with 14C-(U)-D-glucose (5
timates of glutamate specific activity, one sample from jiCi/150 i/l of medium) under three salinity treatments:
each experimental treatment was analyzed by one-di- constant 50% SW (involved transfer between media of
mensional thin-layer chromatography (TLC), as follows. the same salinity), constant 100%. SW, and immediately
Three samples and one lane of dansyl-FAA standards following hyperosmotic transfer from 50% to 100% SW.
(Sigma Chemical) were spotted in four lanes on a 5 X 20 Larvae were sampled attwotime points: 3 h and 6 h after
cm polyamide 6 TLC plate (Baker Chemical). Chro- treatment. After3 hofhyperosmoticstress,concentrations
matogramswereruninachloroform-t-amylalcohol-acetic ofthefive FAAs measured had not increased significantly
acid (70:30:3) solvent system until the solvent front mi- above the 50% SW control (taurine showed a small but
grated 15 cm. Glutamate and aspartate pool sizes were statistically significant drop). Although FAA concentra-
qualitatively assessed under UV illumination, and distri- tions had not yet changed, analysis ofradiotracer incor-
bution ofradiolabel was determined by a 48-72-h auto- poration shows evidence of significant changes in FAA
radiographic exposure. In most cases, the aspartate spot metabolism. Although 80-90% ofall recovered radioac-
was too faint to be detected by eye. Subsequently, chro- tivity in FAA is in the alanine pool under each salinity
matographic regions in the sample lanes corresponding treatment, only proline showed significant variation in
to aspartate and glutamate standards were cut out and specific activity among treatments (Fig. 2). Under either
eluted for scintillation counting. In all tests, the bulk of constant salinity treatment, proline specific activity av-
the label (minimum 75%)wasrecovered in theglutamate
region. Because none ofour qualitative results are signif-
icantly affected by reducing the counts recovered in glu-
GLU
tamate by such a factor, we concluded that pooling the 1
glutamateandaspartatepeaksvia HPLCdidnotintroduce
significant error into the results presented here.
Levels ofFAA measured by HPLC are presented here I
<u
in unitsofnanomoles/larva. The mean (S.E.)wetweight
ofalarvawas 1.37 0.04 mg;dryweightwas0.39 0.02 I
mg. Although the reported values can, therefore, be con- I
vertedto morecommon units(e.g., mmoles/kgtissuewa- f .
ter, or mmoles/g dry weight), the fact that whole mega-
lopas were homogenized would make it difficult to com-
parethevaluespresented here tovaluesreported foradult
tissues. This is because ourwet weights include gut water
content, and our dry weights consist primarily of exo-
skeleton rather than actual FAA-containing tissue.
412 R. S. BURTON
40'
REGULATION OF PROLINE SYNTHESIS 413
414 R. S. BURTON
CHX treatment wasspecifically inhibitorytoproline(but on the proline biosynthetic pathway hasnotyetprogressed
not alanine) synthesis. to thegenetic level among metazoans, available data sug-
gests that homologous gene-enzyme systems are present
(Smith et ai. 1980; Wakabayashi and Jones, 1983).
Discussion
The results presented here for C. sapidus megalopas
Although the role ofproline accumulation in the hy- are similartothose obtained by Burton (1986, 1991b) for
perosmoticresponseisvariableamongtheCrustacea, such thecopepod Tigriopuscalifornicusin suggestingthatpro-
an accumulation of proline is wide-spread among taxa, line synthesis is specifically induced by increases in en-
havingbeen observed amongbacteria (Le Rudulieretal, vironmental salinity ratherthan simply driven by changes
1984), fungi(HoandMiller, 1978),and metaphytes(Bog- in the synthetic rate ofa precursor(i.e., glutamate). Asin
gess et ai, 1976), as well as among marine invertebrates the T. californicus system, induction ofproline synthesis
(FlorkinandSchoffeniels, 1969;Gilles, 1975, 1979;Schof- appears to be dependant on protein synthesis. In both
feniels, 1976). The mechanisms underlying proline ac- systems, the inhibition of protein synthesis with cyclo-
cumulation may includeproteindegradation, uptakefrom heximide decreased proline synthesis and accumulation
the medium, and de novo synthesis. The role of each during hyperosmotic stress but significantly increased al-
mechanism varies among taxa: for example, among mi- anine accumulation. Two possible explanations for the
croorganisms, gram-positive bacteria appear to regulate enhanced alanine accumulation follow: ( 1) By directly
the synthesis or degradation of proline. whereas gram- preventing the incorporation ofalanine and otheramino
negative bacteria achieve accumulation primarily via up- acids into protein, cycloheximide might lead to measur-
take from the medium (Csonka, 1989). able increases in components of the FAA pool. (2) By
There issubstantial variation amongspecieswith regard preventing the induction ofproline synthesis, cyclohexi-
to the importance ofproline in the FAA pool during hy- mide increases theavailability ofalanine precursors(e.g.,
perosmotic response (Claybrook, 1983). Among the glutamate), thereby stimulating alanine synthesis. In T.
Crustacea, proline is insignificant in the FAA pools of californicus, cycloheximide treatment resulted in signifi-
some species but the dominant contributor to the FAA cant incorporation of l4C-label from glutamate into ala-
pool in others. Furthermore, different tissue types vary nine(presumably viaglutamatecatabolism to malateand
dramatically in composition ofthe FAA pool. While pro- then pyruvate, adirectalanine precursor), supportingthe
line is a major contributor to the pool in most adult C. latterexplanation. This effect wasnot observed in C. sap-
sapidus tissues (Gerard and Gilles, 1972), it never ac- idus. so the validity ofthe two hypotheses cannot be re-
counted formore than 25% ofthe FAA pool in seawater- solvedwiththedataavailable. Itshouldbenoted, however,
acclimated animals. In contrast, our data indicate that that ofthe five FAA monitored (glutamate, glycine, tau-
prolineisthepredominant FAA in megalopas, comprising rine,alanine, andproline), onlyalanineshowedincreased
over 50% of the FAA pool. Whether such ontogenetic levels in response to cycloheximide treatment. This sug-
changesinthecomposition ofthe FAA pool arecommon gests that the former hypothesis alone is unlikely to ac-
to other Crustacea has yet to be studied. count fortheobservedpatternofFAAaccumulation when
The regulation ofFAA metabolism in response to os- protein synthesis is inhibited.
motic stress among the Crustacea and other marine in- The similarities between proline regulation in C. sap-
vertebrates is poorly understood. As discussed above and idus megalopas and T. californicus suggest that the in-
in Burton (199la, b), models ofdirect inorganic ioneffects duction ofprolinesynthesisby hyperosmoticstress might
on specific enzymes in FAA metabolism appear to be in- be a common regulatory mechanism among the Crusta-
sufficient toexplain the regulation ofprolinesynthesis for cea. Protein synthesis isclearly required forthe induction
two reasons. (1) Patterns ofincorporation ofradioactive of proline synthesis in both species. While one must be
precursors into proline indicate that rate ofproline syn- cautious about generalizing on the basis ofonly two spe-
thesis is nearly undetectable unless induced by hyperos- cies, our results suggest a need for molecular tools to de-
motic stress (Burton, 1986). (2) Because l4C-labelled glu- termine if the responsible protein is an enzyme in the
tamatewasprovided asa precursorand inhibition ofpro- pathway itself or a regulatory protein of some sort that
tein synthesis prevented proline synthesiswhich occurred stimulates existing enzymes to initiate proline synthesis.
in the absence ofinhibitor, we can conclude that protein
synthesis inhibition acts somewhere in the glutamate to Acknowledgments
proline pathway. Based on available information from
bacteria and yeast, three gene loci encode the enzymes: I thank L. Kordos, J. Bishop, and H. Nguyen for col-
7-glutamyl kinase, -y-giutamyl phosphate reductase, and lecting the crab megalopas, and H. Nguyen for technical
pyrroline-5-carboxylate reductase (Hayzerand Leisinger, assistance. J. Bishop and two anonymous reviewers pro-
1980; Tomenchok and Brandriss, 1987). Although work vided thoughtful commentson the manuscript. Thiswork
REGULATION OF PROLINE SYNTHESIS 415
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