Carbohydrates of the Organic Shell Matrix and the Shell-Forming Tissue of the Snail Biomphalaria glabrata (Say)

10 Pages·1998·4.4 MB·English

by  J C Marxen

Reference; Biol. Bull. 194: 231-240. (Apnl, 1998)
Carbohydrates of the Organic Shell Matrix and the
Shell-Forming Tissue of the Snail
Biomphalaria glabrata (Say)
JULIA C. MARXEN*. MAREN HAMMER. TILMAN GEHRKE, AND WILHELM BECKER
University ofHamburg. Department ofZoology, Martin-Luther-King-Platz 3,
D-20146 Hamburg. Germany
Abstract. Sulfated carbohydrates may play a role in the processes that have to be controlled are the crystal nucle-
biomineralization of the moUuscan shell. The carbohy- ation and then the modification, morphology, and size of
drates of the extracted water-insoluble organic shell ma- the growingcrystals. Macromolecules fromthemolluscan
trix (IM) of the freshwater snail Biomphalaria glabrata water-soluble organic matrix (SM) can modify calcium
were identified as glucose, mannose. galactose, and iV- carbonate crystals in vitro (Belcher et al.. 1996; Falini et
acetyl-glucosamine, whereas the water-soluble organic al, 1996). Furthermore, acidic macromolecules from the
matrix (SM) additionally contained A'-acetyl-galactos- SM can influence the crystal morphology by stereoselec-
amine. A specific lectin binding pattern ofthe matrix was tive binding (Addadi and Weiner, 1985).
obtained. One prominent protein of the SM, with a size The shell-building tissue of molluscs, the mantle, se-
of 19.6 kDa and a pi of7.4, was shown to be a glycopro- cretes all shell material into the extrapallial space below
tein with terminal glucosyl or mannosyl moieties. The the periostracum. Here, the shell is formed in a way that
acidic constitutents of the matrix showed a variety of is still scarcely understood. The mantle edge of pulmo-
possible terminal sugars, indicating a heterogenous mix- nates can be morphologically and physiologically divided
ture ofproteoglycans orglycosaminoglycans (GAGs) and into five zones (Timmermanns, 1969: Bielefeld et al..
glycoproteins. 1993a), which should reflect functional units. Ifthe exact
At the shell-forming mantle edge, an alcian-blue-posi- function of each unit could be defined successfully, the
tive material was observed in the periostracum groove sequence of the functional units would allow us to draw
(PG), the belt, and apically in the cells ofthe outermantle conclusions about the chronology of the mineralization
epithelium (OME). With the help of lectins, all sugars in process. Therefore, it is of special interest to localize the
question were detected in the PG and the belt, whereas production site ofspecific matrix components at the man-
the OME was bound by glucose/mannose- and GlcNac- tle edge.
specific lectins only. Although the complete set ofGAGs In the past, analysis of molluscan shell matrices con-
will be produced in the PG and the belt, a very acidic centrated mainly on the proteins, and minorattention was
fraction of GAGs and the 19.6-kDa protein can also be paid to its carbohydrates. The interest focused primarily
delivered by the OME. on chitin, which is found in the water-insoluble organic
matrix (IM) ofmany molluscs (e.g., Poulicekefa/., 1991)
Introduction
and could play a special role in the structural framework
The biomineralization of the molluscan shell is con- ofthe shell (Weiner and Traub. 1984; Falini etal., 1996).
trolled to a high degree by the organic shell matrix. The But other carbohydrates, especially in the SM, should be
investigated because they might provide sulfate groups,
Received 28 April 1997; accepted 1 December 1997. which possibly concentrate calcium in the shell (Addadi
*To whomcorrespondence should be addressed. E-mail; fb4a010@ et al.. 1987).
rrz-cip-1.rrz.uni-hamburg.de In the SM of several oysters, hexosamines or hexoses
Abbreviarions: GAG, glycosaminoglycan; IM. water-insoluble or-
ganicmatnx; IME,innermantleepithelium; OME,outermantleepithe- and uronic acids are found in combination with sulfate
lium; PG, penostracum groove; SM, water-soluble organic matrix. (Crenshaw, 1972; Samataand Krampitz, 1982). Similarly,
231
232 J. C. MARXEN ET At.
in the SM ofthe aragonitic cross-lamellar structured shell Determination ofalkali-resistant hexosannnes
of the freshwater gastropod Bioinpludaha liUihniici. the Protein was removed by boiling the matrix samples for
amounts of hexosamines, hexoses. uronic acids, and sul- 6 h in 0.5 M NaOH and then washing with 1 M HCl,
fate were 8.8%. 12.7%. 2.1%. and 14% (w/w). respec- 95% (v/v) ethanol. acetone, and 100% ethanol (Jeuniaux,
tively (Marxen and Becker, 1997). After SDS-electropho- 1963). In the alkali-resistant residue, hexosamines were
resis. the SM showed considerable material stained posi- quantified using Ehrlich's reagent as described by Elson
tively with Stains-all and alcian blue. These stained areas and Morgan (1933) and modified by Kabat and Meyer
probably represent glycosaminoglycans (GAGs) bound
(1961).
to proteins. Prominent among the matrix proteins of B.
glahraia was one that, with a size of 19.6 kDa. an isoelec- Infrared spectrometry
trical pointof7.4. and a hydrophobicA'-terminus (Marxen
and Becker. 1997). might be a glycoprotein. A KBr pill of the soluble or insoluble organic matrix
Our approach in this study was the biochemical identi- of B. glabrata was prepared according to the method of
fication of the carbohydrates in the organic shell matrix Giinzlerand Bock (1983), using 1% ofdried organic shell
of B. glahraia. and the detection of glycoproteins. We matrix, and analyzedon aPerkin Elmer841 infrared spec-
expected that the histochemical localization of carbohy- trometer. For a better detection of chitin. protein was
drates at the shell-building mantle edge would reveal removed from some samples of the IM, as described
where the glycosylated components of the organic shell above.
matrix are secreted.
Gas chromatography
Materials and Methods Sugars were identified with gas chromatography (Hew-
Animals pleattckPeadckwairtdh. M3o%deslil4ic3o7nAe)OuVsin2g25a o1n0 mChxromVsoisnocrhbcoWluHmPn
Between 200 and 300 snails ofthe species Biomphala- 80-100 mesh. Samples were prepared according to
ria glabrata (Say. 1818) (Basommatophora. Planorbidae) Chaplin (1982). An optimal methanolysis was achieved
were kept in 80-1 aquaria with a water exchange ofabout with 2M HCl for 16h at 85°C. The dry. methanolyzed
200 1 of dechlorinated tap water per day. The water was samples were dissolved in 500 pX waterfree methanol and
preheated to 28 ± TC, and the illumination cycle was mixed with 10 /jl pyridine and 50 p\ acetic anhydride.
12 h light to 12 h dark. The animals were fed ad libidiim After 5 min ofincubation at room temperature, the sam-
1
with a food prepared according to Standen (1951). ples were dried overnight (Kozulic et ai. 1979). The
reacetylated samples were mixed with 100 /j1 silylation
Extraction ofthe shell matrix reagent (trimethylsilyl imidazole : A',0-bis-(trimethylsi-
lyl)-acetamide trimethylchlorosilane = 3:3:2) (Sweeley
The organic matrix of the shell was extracted as de- et ai. 1963). a:nd incubated for 15 min at 70°C and for
scribed elsewhere (Marxen and Becker. 1997). Briefly: 45 min at room temperature. Inositol was usedas an inter-
100 g ofpowdered shell, iMncluding the periostracum. was nal standard. Injector and detector temperatures were
suspended in 50 ml 10 ' HCl. The shell powder was 250°C. andtheelution program was 2 min at 120°C. rising
decalcified with HCl under continuous stirring at +4°C.
by 6°C/min. to 220°C.
the pH never dropping below 5.0. Each time the volume
reached 250 ml. the pH was adjusted to 7.4 and the prepa-
Lectin binding to the soluble matrix
ration was allowed to rest for 30 min.. then centrifuged
for 20 min. at 16.000 x g. The supernatants were stored, For the visualization of the lectin binding to the SM,
andthepreservatives NaN,and AEBSF(4-(2-aminoethyl) an assay analogous to an ELISA was used. Between all
benzenesulfonyl fluoride) added. The pellet remaining subsequent steps the microliter plate was washed three
after decalcification was washed, lyophilized. and termed times with wash solution (150mW NaCl -I- 0.1%f (v/v)
insoluble matrix (IM). All supernatants from the decalci- Tween 20). 200 ^1/well. A 96-well microtiter plate waMs
fication were combined and dialyzed with 20 changes of coated with SM, each well with 10 pgprotein/50 p\ 0.2
the sixfold volume against bidistilled water. Material that sodium carbonate buffer. pH 9.5. Unspecific binding was
precipitated during dialysis was removed by centrifuga- bk)cked with 200 /vl/well blocking buffer: 1% (w/v) car-
tion at 10^ X g for 30 min. This pellet had an intermediate bohydrate-free BSA in 50 mMTris/HCl + 150 mMNaCl,
degree of solubility in water and was not further investi- pH 7.5. Stock solutionsofthe biotinylated lectins (Vector,
gfautgeadt.ioTnhweavsorleudmueceodf tbhyelsyuoppehrinlaitzaantitonoftoth2e010m'-l,i,a'ncdentfruir-- BIumrgl/inmglamien. 1C0Am)MwePreBSpr+epa1r5e0d minMaNcaoCnlc,entprHati7o.n4 o-fH
ther desalted on a P-2 column (Bio-Rad). The void vol- O.iy/c (w/v) BSA + Q.\% (w/v) NaN,. The lectins ofthe
ume was lyophilized and termed the soluble matrix (SM). stock solutions were diluted 1:100 with dilution buffer:
CARBOHYDRATES OF B. GLABRATA 233
50 rtiM Tris/HCl + 150 niM NaCl, pH 7.5 + 0.5% (w/v) Lectin binding to the isoelectricfocusing gel
B(5S0A^i/+we0l.l)05w%as(v/ivn)cuTbwaeteedn w2i0t.hThtihes lSeMctinfotrest30somliuntioant werWeheapnpltiheed dliErFectrluyntowatshefgienlisahcecd,ordbiiontgintyolaatperdocleecdtuirnes
37°C. Thirty minutes prior to use, the complex of avidin modified from Allen etal. (1976). The gel was fixed with
and biotinylated peroxidase (ABC) (Vector) was pre- 12.5% (v/v) TCA for 15 min and washed three times with
pared; each component was diluted 1:200 in the dilution wash solution (see Lectin binding to the soluble matrix).
buffer. The ABC was incubated 50 /.xl/well for 30 min at The gel was incubated under constant shaking for 1.5 h
3w7e°rCe.iAnscuabaftienadl fsoterp,3050mi/inl/watell37o°fCthiensuthbestrdaatrek.soAluBtTioSn adtilruoteodm1t:e1m0p0erwaittuhre50wimthMlTecrtiisn/sHfCrlom-I-t1he50stmocMk sNoalCult,ionpsH,
(cus2oso,ned2cdi'eu-naamtszriapanteoircbobhionrsr(oao3mt-feoegtt9ehr5nyi.leh3b.yedmnrTgzaht/tehe1i0a+s0zuobm8lsl3itn6reas-mtu6egb-ssbtucuriifatffrtoeiencricabwcuaaifscdfiedmr5)o0nwimnoag-sa 7Azu.osB5le,eC,),,w-I-addsii0ls.uis1tno%elcdvueb(d1av:t/v5ei)n0d DTifnowMretFhe1enh(.sd2aiA0mm,EeeCtahsnoy(dll3uf-wtoaiarmosimnhnaoem3-di09d-metei)htrnhe4yeplmrctgiaio/rmrmbelast,.-o
hydrate -(- 1068 mg disodium hydrogen phosphate dihy- was used as achromogene. The freshly prepared substrate
drate filled up to 100 ml with bidistilled water, pH 4.5. solutioncontained 76 ml of0.05 Macetatebuffer, pH5.0,
The optical densities at 405 nm were measured with a 4 ml of AEC in DMF, and 400/71 3% (v/v) H.O,. The
microplate reader. The quality of all lectins was tested gel was incubated in the dark with the substrate solution
with appropriate neoglycoproteins, which replaced the
until intense red bands appeared.
SM in thesecontrols. The unspecific binding ofthe lectins
to the microliter plate was tested, leaving some weSllMs Histological detection ofmucus
uncoated. The specificity of the lectin binding to the
was tested by preincubating the lectins with suitable car- Pieces ofthe mantle edge were fixed by three methods.
bciofhiycdornaltyeswfhoern30anmiinnh.ibTihteingbienfdfienctg wwaass ocbosnesrivdeedr.ed spe- M(ve/tv)hogdlu1t:arFdiixaaltdieohnydfeori2n80h.0a5tMroocamcotdeymlpaetreatbuurfeferin, 2p%H
7.4. Method 2: Fixation for 24 h at room temperature in
4% (v/v) formaldehyde in 0.067 M phosphate buffer, pH
Lectin binding to the insoluble matrix 7.4, with 0.57r (w/v) cetylpyridinium chloride added.
carTrhieedionuvtestinigsatmiaolnl opflatshtieclcecetnitnribfiungdeinvgialtso.tAhlelIbMuffwearss Mhyedtehoidn p3i:crFiicx-aatciiodn-sfaotrur1a8tehdaett4ha°nColinw4it%h (5v%/v)(vf/ovr)maacledtei-c
and reagents were as described for the lectin binding to atchiidc.kneAsfstewrereembceutd,dianngd tihne pPaarraappllaasstt,wsaesctrioenmsovoefd.5-M/u/-m
the soluble matrix, except that the lectin stock solutions
were diluted 1:200, and the ABC-complex 1:400. with cus and mucus cells were stained with 1% (w/v) alcian
dbuiflfuetri.onTbhufeferv.iaTlshewesraemplfiellseodluctoimopnlwetaesly1 mwgi/thmlbdliolcuktiinogn bsltauiene8dGwXitihne3it%her(vK/ev)meacchettircotacoirdPaAtSp.HFo2.r5thaenddifcfoeurnetnetri--
buffer, kept overnight at 4°C, and then emptied. Next, ation between carboxylic and sulfate groups, the alcian
25 ^1 ofthe constantly stirred sample solution plus 100 ^1 blue staining at pH 1.0 (Lev and Spicer, 1964) and the
ofdiluted lectin was placed in the emptied vials and incu- critical electrolyte concentration (Scott and Doriing,
bated for 30 min at 37°C. The vials were washed three 1965) were carried out.
times with 300 /xl wash solution and centrifuged at 8000
X g for 3 min in between. A 100-^1 sample of the ABC Lectin histochemistry
was incubated and washed in the same way. After incuba- Pieces of the mantle edge were fixed for 26 h at room
tion with 100 /il of substrate solution for 15 min, 50 /j1 temperature in 2% (v/v) formaldehyde in a solution of
was pipetted into a microplate for measuring the optical 25% (v/v) ethanol. 25% (v/v) ethyl acetate. 5% (v/v) ace-
densities at 405 nm. tic acid, and 0.5% (w/v) picric acid. From sections of 7-
/jm thickness, embedded in Paraplast, the Paraplast was
removed, and the endogenous peroxidase was blocked
Isoelectricalfocusing
with 1% (v/v) HjO. in 100% methanol. Unspecific bind-
The isoelectrical focusing (lEF) was performed ac- ing was blocked with 2% (w/v) BSA in PBS (150mM
cording to the recommendations of Serva (Heidelberg, NaCl, buffered with 10 mM phosphate, pH 7.4). The sec-
FRG). Servalyt precotes (125 X 125 mm) with a poly- tions were incubated with biotinylated lectins (Vector
acrylamide layer of 150 //m and a pH gradient from pH Laboratories, Burlingame, CA), in dilutions from 1:50 to
3.0 to 10.0 were used. After 30 min of prefocusing, the 1:1600 in PBS, pH 7.4, containing 0.25% (w/v) BSA and
samples were loaded and separaWted for 1.5 h with a maxi- 0.1% (w/v) NaN, for 18 h at4°C in a moistened chamber.
mum of 2000 V, 6 mA. and 4 at 4°C. After careful rinsing with PBS, the avidin-biotin-peroxi-
234 J. C. MARXEN ET At.
ISOO 1000 600 ISOO 1000 600
frequencyIcrn'M
«X)0 3500 Z500 2000 1500 1000 600 1.000 3S00 3000 ISOO 1000 600
IfequencY[cm-'I frequency[cm'l1
1000 600 ISOO 1000 600
Irequencylcm'l1 frequencyIcm'll
Figure 1. Theinfraredab^o^ption spectrafrom (a)theextractedwater-insolubleorganicmatrix fraction
of the Biomphalaha glabram shell; (b) the alkali-treated, protein-free water-insoluble organic matrix; (c)
chitin from crabshell; (d) the extracted water-soluble organic matrix fraction of the B. glabnmi shell; (e)
mucin from bovine submaxillary glands; (f) chondroitin sulfate A from bovine trachea.
dase complex (Vector) was incubated for 30 min at room [w/w]), 3.4% (w/w) was composed ofhexosamines. After
temperature. After rinsing with PBS. the staining was a previous alkaline treatment, alkali-res—istant hexosa-
carried out with 0.08% (w/v) DAB, 0.075% (w/v) NiCl.. mines represented 2.9% (w/w) of the IM that is, 85%
and 0.01% (v/v) HjO. in Tris-buffered solution, pH 7.4, (w/w) of the total hexosamines. The hexosamines of the
for 20 min at room temperature. Controls: ( 1) without SM were not alkali-resistant.
lectin, (2) without ABC, (3) without DAB, (4) lectins The finding of alkali-resistant hexosamines in the IM
preincubated with their specific sugar. hinted at the occurrence ofchitin. The infrared absorption
spectra of the IM and SM of the Bioniphalaria glahrata
Results shell (Fig. 1) were examined to see whether chitin was
visible in the IM and whether the occurrence of GAGs
Hexosamine quantification and infrared spectrometry in the SM could be confirmed. The main absorption bands
In this preparation, the IM of B. glabrata included the are listed in Table I.
periostracum. Ofthe hydrolyzable part of the IM (63.5% The pattern of the IM (Fig. la) differed from that of
CARBOHYDRATES OF B. GLABRATA 235
Table I
Positions ofthe main infraredabsorption hands ofthe extracted water-soluble organic matrixfraction ofthe Biomphalaria glabrata shell (SM).
mucin from bovine submaxdlaryglands, chondroitin sulfateAfrom bovine trachea, the water-insoluble matrixfraction (IM). the alkali-lrealed
protein-free IM. andchitinfrom crabshell: appropriate functional groups are suggested in the left column
236 J. C. MARXEN ET AL.
Table III
The bindingpattern oflectins to tlie extracted water-soiidile (SM) and insotulile (IM) organic matrix fractions oftlie Biomphalaria glabrata sliell
Lectin
CARBOHYDRATES OF B. GLABRATA 237
positive components of the periostracum of B. iilaivaia.
but the amount may be considered rather low.
Glycoproteins und proteoglycans
Prominent among the proteins ofthe SM ofB. glahrata
is one that has a size of 19.6 kDa and an i.soelectrical
point of 7.4. A'-terminal microsequencing revealed that
15 or 16 ofthe 24 amino acids identified in the 19.6-kDa
protein were hydrophobic (Marxen and Becker. 1997).
Because of its high pi, this protein cannot be directly
involved in the binding of calcium. As demonstrated by
the binding of lectins to the lEF gel (Fig. 2, Table IV),
this protein is glycosylated with glucosyl and mannosyl
moieties, singly or in combination. Thus, this glycopro-
tein contains hydrophobic as well as hydrophilic domains
and may have evolved from a membrane protein. In the
SM ofMytUiis I'dulis. Keith cl al. (1993) found a protein
with a size of21 kDa and a highly hydrophobic A'-termi-
nus with a .sequence that was identical in the positions 7,
8. and 9 to that of the 19.6-kDa protein of B. glahrata.
It is not known, however, whether the 21-kDa protein
from M. edulis is glycosylated. Mann et al. (1988) ob-
served a change in the modification ofcalcium carbonate
crystals under a stearic acid monolayer. The hydrophiibic
and hydrophilic parts of the 19.6-kDa protein (and per-
haps also the 21-kDa protein from M. edulis) could give
molecules o—f this kind a detergent-like quality, —by which
ihey might among other possible functions play a
role in the determination of the crystal modification.
The acidic material of the SM of B. glahrata shows
a variety of possible terminal sugar moieties at various
isoelectrical points (Fig. 2. Table IV). Although the main
Figure 2. Isoelectrical focusing of the extracted water-soluble or-
ganicmatrixfractionoftheBiomphalariaglahratashellandapplication part with a broad range of isoelectrical points is bound
oflectins. Std = standard proteins; Coom = matrix, stained with Coo- by the Gal- or GalNac-specific MPA, only a very acidic
massie brilliant blue; ConA. WGA. and MPA = lectins: Alz = matrix, component is detected by the GlcNac-specific WGA. A
stained with alcian blue. lower acidic part is detected by ConA, pointing to Man or
Glc, which are not common sugars in GAGs. The results
indicate the occunence ofseveral different GAGs and gly-
the belt. The calcium cells in the interstitiiim bound al- coproteins. Mixtures of GAGs are common in vertebrates
(Volpi, 1996) as well as in molluscan tissues (Dietrich et
omcocsutrraeldl,leicntdiincsa,tibnugt mneoreilnyhibaintiuonnspbeycitthiec srpeeaccitfiiocn.sugars al.. 1983). Cottrell et al. (1994) detected a large number of
hexoses and hexosamines in th—e body mucus of the slug
{Ariou ater) and showed that in additi—on to the main
Discussion fracfion, which probably is heparan sulfate other, uniden-
tified GAGs unknown in vertebrates must also be present.
Chitin
Moreover, in invertebratesthe variable glycosylation ofone
The hexosamines of the IM were mainly alkali-resis- core protein is possible (Har-El and Tanzer. 1993).
tant, so the matrix could contain chitin. The infrared spec- Mucopolysaccharides have been found in other mol-
trometry could not, however, confirm this assumption luscan shells as well (Simkiss, 1965; Worms and Weiner,
(Fig. lb). Bielefeld cl al. (1993a), using electron micros- 1986), buttheirfunction inthe shell remainsquestionable.
copy, found WGA binding sites in the periostracum of Sulfated polysaccharides have been discussed as possible
B. glabrata. After a chitinase digestion, the reactivity of calcium-binding sites (Wilbur, 1976) and, becau.se oftheir
these sites was reduced, but not negative. The cells at the appearance in the center of nacreous tablets, could play
PG and the belt, however, were not affected by chitinase. a role in the nucleation and the growth inhibition of the
The results indicate that chitin is one of the GlcNac- mineral (Crenshaw and Ristedt, 1976).
238
CARBOHYDRATES OF B. GLABRATA 239
All the glycosylated components of the matrix can be
produced in cells of the PG (Zone 1) and the distal belt
(Zones 2 and 3). In contrast to Zones 1 to 3, the proximal
OME
belt (Zone 4) and the (Zone 5) exhibitedonly termi-
nal GlcNac and Man/Glc, respectively pointing to the
production of a GAG with a pi of 3.5 and to the less
acidic glycosylated material, the 19.6-kDa glycoprotein,
or both. The lectin binding pattern of the mantle edge
kinidnidcsatoefsGaAGfsun.ctbiuotnatlhedsiaffmeereGntAiaGtihonasadmifofnegrentthefuvnacrtiioounss epiFtihgeulrieum6.oiBBiionmdpihnaglaorfiathgelalberctaitna.CTohneApo(s1i:t2i0v0e)rteoactthieonouptreordumcatnctalne
depending on the location of its production. be seen apically (small arrow); the gray appearance of the cell bodies
is caused by their natural orange color (large arrow).
Conclusions
The striking difference in the lectin binding pattern (Wilbur, 1964; Watabe, 1984). Because calcium has also
between the distal part (Zones 1 to 3) and the proximal been localized at these proximal zones in B. glabrata
part (Zones 4 and 5) ofthe shell-forming tissue gives new (Bielefeld et al.. 1992), this site can be considered to be
emphasis to a strict functional separation between these the region where the calcification of the organic matrix
parts. of the shell takes place.
In Zones 1, 2, and 3 of the mantle edge of freshwater The periostracum is produced by the groove and the
snails, a phenol oxidase activity has been observed (Tim- distal belt (Bielefeld et al.. 1993a). Furthermore, the belt
mermanns, 1969; Bielefeld et al.. 1993a). This enzyme seems to be the production site for those GAGs and pro-
may be responsible for the sclerotization and tanning of teins that form the structural framework of the organic
the periostracum (Waite, 1984) and the matrix (Gordon matrix. Here, the proteins will be partly linked by the
and Carriker, 1980), which thus become water-insoluble. phenol oxidase, trapping acidic polysaccharides. Matrix
The sugar patterns in the SM and IM of B. glabrata are constituents that may be directly involved in the calcifica-
very similar, indicating that GAGs are trapped in the tion process ofthe shell seem to be produced, in addition,
network of sclerotized proteins. by the mineralizing region of the mantle. In B. glabrata.
In Zones 4 and 5, a strong alkaline phosphatase activity these constituents presumably include the very acidic
(Timmermanns, 1969; Bielefeld et al.. 1993b) and a car- WGA-positive part of the GAGs, the less acidic ConA-
bonic anhydrase activity were detected (Timmermanns, positive material, andthe 19.6-kDaprotein. Becausecom-
1969; Boer and Witteveen, 1980); both enzymes are ponents of the SM are known to enhance crystal nucle-
thought to be closely related to the mineralization process ation when immobilized but inhibit crystal growth when
free in solution (e.g., Wheelerand Sikes. 1989), the func-
tion of the acidic polysaccharides may vary depending
«&••* '^* >-';|i GonAGthseirmapylacperoovfidoeringuicnl.eaDtiisotanllsiyteps,rowdhuicleed,theimpmroobxiilmiazleldy
f produced ones could instead be involved in regulating
crystal growth.
i»; • .--*«•) /i^'c?-/<
m Acknowledgments
'.'I
We thank Mrs. V. Wagschal forher excellent technical
assistance. This project was financially supported by the
German Ministry for Research and Technology (BMFT)
C* WB
(50 9112).
« V ,..'" WE
•• I
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