Characterization of the Proteins Comprising the Integral Matrix ofStrongylocentrotus purpuratus Embryonic Spicules*

(Received for publication, October 17, 1995)

Christopher E. Killian and Fred H. Wilt‡

From the University of California, Berkeley, Department of Molecular and Cell Biology, Division of Cell and DevelopmentBiology, Life Sciences Addition, Berkeley, California 94720-3200

In the present study, we enumerate and characterizethe proteins that comprise the integral spicule matrix ofthe Strongylocentrotus purpuratus embryo. Two-dimen-sional gel electrophoresis of [35S]methionine radiola-beled spicule matrix proteins reveals that there are 12strongly radiolabeled spicule matrix proteins and ap-proximately three dozen less strongly radiolabeled spic-ule matrix proteins. The majority of the proteins haveacidic isoelectric points; however, there are several spic-ule matrix proteins that have more alkaline isoelectricpoints. Western blotting analysis indicates that SM50 isthe spicule matrix protein with the most alkaline iso-electric point. In addition, two distinct SM30 proteinsare identified in embryonic spicules, and they have ap-parent molecular masses of approximately 43 and 46kDa. Comparisons between embryonic spicule matrixproteins and adult spine integral matrix proteins sug-gest that the embryonic 43-kDa SM30 protein is an em-bryonic isoform of SM30. An adult 49-kDa spine matrixprotein is also identified as a possible adult isoform ofSM30. Analysis of the SM30 amino acid sequences indi-cates that a portion of SM30 proteins is very similar tothe carbohydrate recognition domain of C-type lectinproteins.

During the course of its development a sea urchin embryoconstructs a pair of calcareous endoskeletal spicules. Thesespicules are rod shaped, mineralized structures which are cal-citic assemblages of calcium carbonate (95%) and magnesiumcarbonate (5%) with an occluded proteinaceous integral matrix.The spicules are synthesized by a well characterized singletissue type, the primary mesenchyme cells. The calcite of eachspicule rod is aligned along a single crystal axis, appearing asif each spicule is composed of one crystal of calcite. However,the spicule has greater flexural strength than a single crystal ofcalcite and it fractures as if it is made up of many microcrystals(1–3). Persuasive biophysical evidence indicates that the pro-teins embedded within the mineral phase of the spicules, theintegral spicule matrix proteins, cause these interesting phys-ical characteristics (1, 4–7). It is believed that these proteinsinteract with specific faces of the calcite crystal when occludedwithin the mineral, and it is through these interactions thatcontrol of spicule growth occurs. However, the precise molecu-

lar mechanisms underlying interactions with noncollagenousintegral matrix proteins that control the formation and thephysical properties of mineralized tissues remain unknown. Aquestion basic to our understanding of these mechanisms inmineralizing tissues is what is the nature of the noncollag-enous integral matrix proteins that are intimately associatedwith the mineral portion of these tissues.Many proteins have been identified and characterized as

noncollagenous integral matrix proteins of hard tissues fromvarious vertebrate and invertebrate organisms (most numer-ously from vertebrate bone and teeth). These types of proteinsusually share the general properties of being soluble and acidic(8, 9). However, it has proven difficult to ascribe to these typesof proteins precise functions within the cell that synthesizethem. In addition, while many noncollagenous integral matrixproteins have been identified, it has also proven difficult todetermine with much certainty what particular integral matrixproteins are contained within a given mineral phase at a givenstage of development. Many of these difficulties are due to thecomplex structures and dynamics of the mineralized tissuesmost widely studied, i.e. vertebrate bone and teeth.Sea urchin spicule formation, on the other hand, is particu-

larly well suited to ask these sorts of basic questions. Thespicules are synthesized by a single well characterized tissuetype and they are relatively simple mineralized structures thatdo not have the complex dynamics of vertebrate bones or teeth.The sea urchin embryo is also very amenable to biochemicaland molecular experimental analysis. Much is known about thecell and developmental biology of sea urchin embryos and par-ticularly about the differentiation of the cell lineage whichsynthesizes the spicules (for some reviews, see Refs. 10–14).Benson et al. (15) reported that the spicule matrix within themineralized spicules is arranged in concentric sleeves of irreg-ular fibrillar proteinaceous material with some interconnec-tions between the layers of matrix material. The concentriclayers of the spicule matrix are also reflected in the concentriclayered architecture of the mineralized sea urchin spicules (2).Benson et al. (16) and Venkatesan and Simpson (17) also iden-tified 8–10 different proteins as comprising the integral spiculematrix. In these studies, one-dimensional SDS-PAGE1 wasused to resolve the spicule matrix proteins. In addition, bothreports demonstrated that most of these detected proteins areN-linked glycoproteins. Total amino acid analysis revealedStrongylocentrotus purpuratus spicule matrix proteins are richin acidic amino acids (16). This amino acid composition issimilar to that of other integral matrix proteins closely associ-ated with the mineral portion of other mineralized tissues(8, 9).Two different cDNAs that encode two different spicule ma-

* This work was supported by National Institutes of Health GrantHD 15043 (to F. H. W.) and National Aeronautics and Space Adminis-tration Grant NAG 5–72 (to F. H. W.). The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.‡ To whom reprint requests and correspondence should be sent: Uni-

versity of California, Berkeley, Dept. Molecular & Cell Biology, LifeSciences Addition, Rm. 379, Berkeley, CA 94720. Tel.: 510-642-2927;Fax: 510-643-6791; E-mail: ckillian@uclink2.berkeley.edu.

1 The abbreviations used are: PAGE, polyacrylamide gel electro-phoresis; CRD, carbohydrate recognition domain; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium chloride.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 15, Issue of April 12, pp. 9150–9159, 1996© 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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trix proteins have also been isolated from S. purpuratus cDNAexpression libraries. The first cDNA isolated encodes a proteindesignated SM50 which has a deduced amino acid sequencewith a molecular mass of approximately 50 kDa (18–21). Thesecond cDNA cloned encodes a protein designated SM30 whichhas a derived amino acid sequence with a molecular mass ofapproximately 30 kDa (22). The predicted chemical character-istics of the deduced amino acid sequences of SM50 and SM30cDNAs are somewhat different. The cloned SM50 cDNA en-codes a protein with an alkaline pI without any consensusN-linked glycosylation site (21). The cloned SM30 cDNA en-codes an acidic protein that contains a consensus N-linkedglycosylation site (22). Both SM50 and SM30 transcripts areexpressed exclusively in the primary mesenchyme cells (19,22). Sucov et al. (18) have shown that SM50 is a single copygene in S. purpuratus. Alternatively, there is experimentalevidence that there is a small family of SM30 protein genes.Akasaka et al. (23) presented Southern blotting analysis indi-cating that there are between two and four copies of SM30genes present in the S. purpuratus haploid genome. Akasaka etal. (23) further demonstrated that an isolated S. purpuratusgenomic clone contains two different SM30 genes that arearranged tandemly. These two SM30 genes were designatedSM30-a and SM30-b. Initial characterization of the genomicregulatory regions of the SM50 gene and the SM30-a gene havealso been done (23–26).In addition to these two genes that have been shown directly

to encode two spicule matrix proteins, a recent report by Har-key et al. (27) characterizes a gene encoding a nonglycosylated27-kDa protein, designated PM27, that is closely associatedwith growing sea urchin spicules. While they did not showdirectly that the PM27 protein is an integral spicule matrixprotein, they do show PM27 expression and biochemistry aresimilar to what one might expect from a spicule matrix protein.In addition they show that PM27 has some sequence similarityto SM50; Harkey et al. (27) point out that portions of PM27,SM50, and the Lytechinus pictus homologue of SM50 (desig-nated LSM34 by Livingston et al. (28)) also have some similar-ity to the carbohydrate recognition domain (CRD) of a numberof C-type lectin proteins.The present paper enumerates more accurately the complex-

ity of the S. purpuratus spicule matrix proteins and more fullycharacterizes these proteins. These studies provide a biochem-ical foundation important for the study of the noncollagenousintegral matrices of mineralized tissues. Our findings also com-plement the previously mentioned biophysical studies that ex-amined occluded matrix proteins of sea urchin embryonic andadult mineralized structures and their roles in regulating min-eralized tissue formation and structure (1, 4–7). The studies inthe present paper reveal that there are 12 spicule matrix pro-teins that radiolabel intensely with [35S]methionine and ap-proximately three dozen other spicule matrix proteins that areless highly radiolabeled. The majority of the spicule matrixproteins have an acidic pI, while several other moderatelyradiolabeled to less radiolabeled spicule matrix proteins have amore alkaline pI. Polyclonal antisera that react specificallywith the proteins encoded by the previously cloned SM50 andSM30 spicule matrix cDNAs were generated. Western blottinganalysis using these antisera identify the SM50 and SM30proteins. In addition, comparisons are made between the em-bryonic spicule matrix proteins and the adult spine integralmatrix proteins. Further analysis of the protein encoded bySM30-a reveals that a portion of the SM30 proteins is similarto the CRD of the C-type lectin family of proteins.

EXPERIMENTAL PROCEDURES

Culturing of Sea Urchin Embryo—S. purpuratus gametes were col-lected, eggs were fertilized, and embryos cultured as described byGeorge et al. (22).Isolation of Spicule Matrix and Spine Matrix Protein—Unlabeled S.

purpuratus embryonic spicule matrix proteins were isolated essentiallyas described by Venkatesan and Simpson (17) except that, as a finalstep, spicules were incubated in 3.5% sodium hypochlorite and thenwashed extensively with water before they were demineralized with 0.5N acetic acid. After the calcite was dissolved, the acetic acid was neu-tralized with Tris base and the spicule matrix proteins were extensivelydialyzed against dH2O. Proteins were then concentrated by lyophiliza-tion. Adult S. purpuratus spine integral matrix proteins were isolatedas described by Richardson et al. (29).Radiolabeled S. purpuratus spicule matrix protein was isolated from

micromeres cultured in seawater with 4% horse serum and [35S]methi-onine. The isolation and culture of micromeres was done essentially asdescribed by Benson et al. (30). The micromeres isolated from about 2 3106 16-cell embryos were cultured in four 100-mm Petri plates, eachcontaining 10 ml of seawater containing 4% horse serum that had beendialyzed against seawater. Two hundred mCi of [35S]methionine (1000Ci/mmol; Amersham) were added to each plate just prior to the onset ofspiculogenesis and left in the medium until the time of harvest (24–72h). At the conclusion of labeling, carrier spicules from whole embryoswere added to the cultures, and the adherent spicules of the culturewere scraped from the Petri plates. The spicules were washed with andthen placed into 3.5% sodium hypochlorite overnight at room temper-ature. They were then washed with 5–7 changes of dH2O. After the finalwash, the spicules were suspended in 1 ml of dH2O. An aliquot wasremoved to quantitate the amount of radioactivity incorporated intospicule matrix. To prepare the radiolabeled spicule matrix protein foreach two-dimensional gel, 5.0 3 104 dpm of the radiolabeled spiculesample was trichloroacetic acid precipitated with 10 mg of cytochrome ccarrier; this procedure dissolves the calcite and precipitates the spiculematrix protein. The pellet was then washed with acetone to removeresidual trichloroacetic acid. The pellet was dried and dissolved in theappropriate sample buffer.Isolated spicule matrix proteins were labeled in vitro with biotin

following the protocol described by Meier et al. (31) using the biotiny-lation agent NHS-CC-biotin purchased from Pierce. The labeled pro-teins were localized using an enhanced chemiluminescence protocoldescribed by Nesbitt and Horton (32) with the exception that the block-ing agent used was 0.1% fish gelatin (Amersham), 0.8% bovine serumalbumin, 0.02% Tween 80, 10 mM Tris, pH 8.0, 100 mM NaCl, and thedilution of strepavidin-horseradish peroxidase (Amersham) used was1:3000.Glycosidase treatment of spicule matrix protein was carried out at

37 °C overnight using endoglycosidase F/N-glycosidase F purchasedfrom Boehringer Mannheim following the protocol provided by themanufacturer.Mild alkaline hydolysis b-elimination of O-linked carbohydrate moi-

eties on spicule matrix proteins was done essentially as described byFlorman and Wassarman (33). Spicule matrix proteins were incubatedin 5 mM NaOH for 24 h at 37 °C, neutralized with HCl, and thenanalyzed by Western blotting analysis as described below. Serine andthreonine O-glycosidic linkages are known to be labile in alkalineconditions (34).Two-dimensional Gel Electrophoretic Separation of Spicule Matrix

Proteins—Two-dimensional gel electrophoresis of spicule matrix pro-teins was carried out using a Bio-Rad mini-protean II two-dimensionalgel apparatus. The protocol followed was essentially that described bythe gel apparatus manufacturer which is based on the protocol ofO’Farrell (35). Pharmolyte ampholytes with pH ranges of 2.5–4.5, 4.0–6.5, and 3–10 were used (Pharmacia). The first dimensions of the gelsshown in Figs. 2, A and B, and 6A were run in the acidic direction usinga blend of equal amounts of pH 2.5–4.5 and 4.0–6.5 ampholytes. Thefirst dimensions of the gels shown in Figs. 2C and 6B were run in thebasic direction using pH 3–10 ampholytes. The nonequilibrium pHgradient gels (first dimensions for Figs. 2, A and C, and 6B) were run at750 V for 20 min. The equilibrium isoelectric focusing gels (first dimen-sion for Figs. 2B and 6A) were run at 750 V for 2 h. The seconddimensions of all two-dimensional gels and all one-dimensional gelswere 10% acrylamide SDS gels as formulated by Laemmli (36) andmodified by Dreyfus et al. (37). Two-dimensional gels containing radio-labeled protein were prepared for fluorography as described by Laskeyand Mills (38).Generation of Polyclonal Antisera—Fusion proteins were engineered

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and used as immunogens for the generation of polyclonal antiseraspecific for the proteins encoded by the previously cloned SM50 andSM30 cDNAs. These fusion proteins were generated by subcloning thecDNAs into the maltose-binding protein expression vector pMal-cRI(New England BioLabs). The 1.3-kilobase lgt11 cDNA clone, pHS72,which encodes a truncated SM50 protein (168 amino acids of the car-boxyl end of the protein) (18, 21), was subcloned into the EcoRI site ofthe pMal-cRI vector. In addition, the 1.8-kilobase pNG7 lgt11 cDNAclone, pNG7, which encodes a complete SM30 protein (22), was alsosubcloned into the EcoRI site of pMal-cRI. These engineered fusionprotein plasmids were then used to transform XL-1 Escherichia coli(Stratagene). The induction of these fusion constructs, the lysis of theexpressing bacteria, and the enrichment of the fusion proteins by affin-ity chromatography using amylose resin were done as described by theaccompanying protocol provided by New England BioLabs. The onlydeviation was that the bacteria harboring the SM30 maltose-bindingprotein fusion were grown at 30 °C instead of 37 °C. This was done toprevent the SM30 fusion protein from becoming insoluble. The SM50and SM30 fusion proteins were then used as immunogens in rabbits togenerate polyclonal antisera following the protocol described by Harlowand Lane (39). The anti-SM30 antiserum was treated with ammoniumsulfate and the immunoglobin fraction was collected and dialyzed asalso described by Harlow and Lane (39). The anti-SM50 antiserum wasused without further treatment.A rabbit polyclonal antiserum raised against all of the spicule matrix

proteins was generated following the procedure described by Benson etal. (16) and using spicule matrix protein isolated from embryonic spic-ules as immunogen.Western blotting of one- and two-dimensional gels was done as de-

scribed by Towbin et al. (40). Chemiluminescent detection of immuno-reactive proteins was done following the directions of the manufacturer(Amersham) except that 0.1% fish gelatin (Amersham), 0.8% bovineserum albumin, 0.02% Tween 80, 10 mM Tris, pH 8.0, 100 mM NaCl wasused as the blocking solution. The anti-SM30 antiserum was used at a1:2000 dilution and the anti-SM50 antiserum was used at a 1:1000dilution for the chemiluminesence blots.Detection of immunoreactive proteins using alkaline phosphatase-

conjugated secondary antibody and 5-bromo-4-chloro-3-indolyl phos-phate/nitro blue tetrazolium chloride (BCIP/NBT) as substrate wasdone as described by Richardson et al. (29). The anti-total spiculematrix antiserum was used at a 1:1000 dilution, the anti-SM30 anti-serum was used at a 1:250 dilution, and the anti-SM50 antiserum wasused at a 1:100 dilution for these blots.In Vitro Translation of SM30 RNA by Xenopus Oocytes and Reticu-

locyte Lysate—The 1.8-kilobase full-length pNG7 SM30 cDNA (22) wassubcloned into the EcoRI site of pGEM4Z (Promega). This resultingplasmid was then used as a template to synthesize capped in vitroSM30 RNA using the Ambion Megascript kit by following the protocolprovided by Ambion.The microinjection of the synthesized RNA into Xenopus oocytes and

the collection of [35S]methionine-labeled secreted proteins was done asdescribed in Livingston et al. (28). The immunoprecipitation of [35S]me-thionine-labeled proteins using the anti-SM30 antiserum or its preim-mune serum at a dilution of 1:100 was also done as described byLivingston et al. (28). SM30 RNA was translated by reticulocyte lysateusing the Ambion Reticulocyte Lysate kit following the protocol pro-vided by Ambion.

RESULTS

Enumeration of the Spicule Matrix Proteins—To accuratelyenumerate the proteins comprising the occluded integral ma-trix of the S. purpuratus sea urchin spicules, spicule matrixproteins were separated using high resolution two-dimensionalgel electrophoresis. Given their very acidic makeup, the spiculematrix proteins do not stain very strongly with conventionalprotein stains such as Coomassie and silver stains. To get areasonable silver staining signal of spicule matrix proteins on aone-dimensional SDS-PAGE required loading a relatively largeamount of protein. This same amount of protein loaded onto atwo-dimensional gel resulted in excessive streaking of many ofthe spicule proteins. We also saw this excessive streaking ontwo-dimensional gels loaded with isolated spicule matrix pro-teins that were labeled in vitro with biotin and then subse-quently localized using strepavidin-horseradish peroxidasebased chemiluminescence. The streaking, we assume, is caused

by overloading of the gel and/or the natural tendency of glyco-proteins to streak on two-dimensional gels (41). This occur-rence makes it hard to enumerate reliably the different spiculematrix proteins (data not shown).We decided, alternatively, that radiolabeling the proteins

with [35S]methionine was a better way to visualize these pro-teins. We found culturing isolated micromeres in the presenceof [35S]methionine to be the most effective method of labelingsea urchin spicule matrix proteins. Harkey and Whiteley (43)demonstrated that the time course of spicule formation in S.purpuratus micromere cultures closely matches the timecourse of events in whole embryos and that the two-dimen-sional gel pattern of proteins synthesized by cells from micro-mere cultures closely matched that of primary mesenchymecells in intact sea urchin embryos.The radioactively labeled spicule matrix proteins synthe-

sized in culture were isolated and then fractionated by gelelectrophoresis. A typical one-dimensional gel fractionation ofthese radiolabeled proteins is displayed in Fig. 1A. This patternis very similar to the pattern we observed for spicule matrixproteins isolated from whole embryo spicules that were labeledin vitro with biotin. The pattern of biotinylated proteins isdisplayed in Fig. 1B. The most noticeable differences in theradiolabeled and biotinylated spicule matrix patterns is that aband at approximately 27 kDa is more prevalent on the radio-labeled protein gel and there is strongly labeled material at$200 kDa on the biotinylated protein gel. The one-dimensionalSDS-PAGE patterns observed here are very similar to thepatterns shown by one-dimensional SDS-PAGE silver stainingand radiolabeling spicule matrix preparations of whole em-bryos that were observed by Benson et al. (16) and Venkatesanand Simpson (17). These previously published one-dimensionalSDS-PAGE patterns did not have the prominent bands at $200kDa that is present on the biotinylated spicule matrix SDS-PAGE pattern. Taken together these results indicate that ra-diolabeling spicule matrix proteins from micromere culturesgenerates a labeling pattern similar to patterns obtained withstains. Furthermore, given its greater sensitivity and clarity,we found radioactive labeling the spicule matrix proteins betterfor further two-dimensional gel analysis.The results of two-dimensional gel analysis of the radiola-

beled spicule matrix proteins are shown in Fig. 2. In Fig. 2A,

FIG. 1. One-dimensional SDS-PAGE of radiolabeled and bioti-nylated spicule matrix protein. A, [35S]methionine radiolabeled spi-cule matrix proteins were separated by a 10% SDS-PAGE gel. Theradiolabeled proteins were then localized using fluorography. B, bioti-nylated spicule matrix proteins were separated on a 10% SDS-PAGEgel and then electroblotted to nitrocellulose membrane. The biotiny-lated proteins were then localized on the membrane using an enhancedchemiluminescence protocol described under “ExperimentalProcedures.”

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the spicule matrix proteins are separated in the first dimensionon the basis of their pI using a non-equilibrium pH gradient gelin which the proteins migrate in the acidic direction for a shorttime (about 20 min). The pH range of the ampholytes used inthis gel was between 2.5 and 6.5. In Fig. 2B, the spicule matrixproteins were again separated in the first dimension on thebasis of their pI, using the same pH range of ampholytes as inFig. 2A. However, in this gel, the proteins were allowed tomigrate to their equilibrium pI. In Fig. 2C, the proteins werealso separated on the basis of their pI in the first dimension.

The pH range of the ampholytes used here, however, was 3 to10, and the first dimension was run in the opposite, basicdirection for only short time (about 20 min), which does notallow the proteins to migrate to their equilibrium pI. If theseproteins were allowed to migrate to their equilibrium pI, themost alkaline proteins would migrate off the alkaline end of thegel.It is apparent most of the proteins are acidic, with molecular

masses ranging from about 20 to over 100 kDa. The 8–10 bandspreviously identified by Benson et al. (16) and Venkatesan andSimpson (17) as S. purpuratus spicule matrix proteins haveapparent molecular masses similar to many of the proteinsseparated by the two-dimensional gels presented here. All ofthe previously identified bands can be accounted for by proteinsfractionated on the gels presented in Fig. 2. Fig. 2, A and B,show that the majority of spicule matrix proteins, as well as allof the most highly radiolabeled proteins, are indeed acidic. Fig.2A reveals a bit more complexity of the matrix proteins thanseen in Fig. 2B, especially for the proteins migrating betweenapparent molecular masses of 30 and 69 kDa. This result isprobably because the most heavily radiolabeled proteins on Fig.2A do not streak out as much before they reach their isoelectricpoint as they do in Fig. 2B. Since most of the spicule matrixproteins are glycoproteins, one expects to see streaking onequilibrium gels (41). This streaking presumably occurs be-cause of the heterogeneity in the charge provided by the car-bohydrate moiety. Since the first dimension of the gel in Fig. 2Bhas been run to equilibrium, it provides a more accurate rela-tive comparison of the pI values of the various spicule matrixproteins.The gel shown in Fig. 2C resolves spicule matrix proteins

that have a more alkaline pI. There are a number of spiculematrix proteins in the more acidic portion of the gel in Fig. 2Cthat also resolved in the more alkaline portion of the gels inFig. 2, A and B. The proteins labeled I, II, and III (Figs. 2 and3) were used as landmarks to orient the proteins in all three ofthe gels. Fig. 2C demonstrates that there are several spiculematrix proteins, with medium to low radiolabeling, which havea somewhat more alkaline pI. The drawing in Fig. 3 is arepresentation of the distribution of the various spicule matrixproteins combining the results from the three kinds of gelspresented in Fig. 2. While the designation of relative abun-dance is problematic with radiolabeled proteins, it is apparentfrom the three gels shown in Fig. 2 that there are 12 spiculematrix proteins that are more highly radiolabeled by [35S]me-thionine than the rest. These proteins are designated in Fig. 3with stars. Given that the most highly radiolabeled spiculematrix proteins have molecular weights coincident with themost highly staining bands of the one-dimensional SDS-PAGEpatterns of spicule matrix proteins shown in Fig. 1B and inBenson et al. (16), and Venkatesan and Simpson (17), thissuggests that the highly radiolabeled spicule matrix proteinsmay actually be some of the more prominent spicule matrixproteins.Fig. 2, A-C, also shows approximately three dozen spicule

matrix proteins that radiolabel less prominently and they arealso represented in Fig. 3. We believe that these less highlyradiolabeled proteins are not contaminants since we get verysimilar patterns on two-dimensional gels with many differentbatches of radiolabeled spicule matrix proteins. The only dif-ferences between batches that we see regularly are slight vari-ations in the relative signals among the various proteins. Thereis other evidence that the radiolabeled proteins we have iden-tified are not contaminants of non-spicule matrix proteins.Benson et al. (16) using scanning electron microscopy saw thatbleach-treated spicule are completely free of embryonic and

FIG. 2. Two-dimensional gel electrophoresis of radiolabeledsea urchin spicule matrix proteins. [35S]Methionine radiolabeledspicule matrix proteins were separated by two-dimensional gel electro-phoresis. The first dimension separates the proteins on the basis oftheir pI using different conditions for each of the three gels. The seconddimension of all three gels is a 10% SDS-PAGE gel. A, the first dimen-sion of this gel was a nonequilibrium pH gradient tube gel usingampholytes with a pH range of 2.5 to 6.5. The gel was run in the acidicdirection for 20 min before placing it on the second dimension. B, thefirst dimension of this gel is an isoelectric focusing gel using ampholyteswith a pH range of 2.5 to 6.5. The gel was run in the acidic directionuntil the proteins came to equilibrium at their pI (about 2 h), beforeplacing it on the second dimension. C, the first dimension of this gel wasa nonequilibrium pH gradient gel using ampholytes with a pH range of3 to 10. The gel was run in the basic direction for 20 min before placingit on the second dimension. After all the gels were run in both dimen-sions, the gels were processed for fluorography and exposed to x-rayfilm. I, II, and III labels indicate the three spicule matrix proteins thatwere used to align the proteins that were common among the gels inA-C. NEPHGE, non-equilibrium pH gradient gel electrophoresis.

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blastocoelic contaminants. In addition, we found that the two-dimensional pattern of radiolabeled spicule matrix proteinsisolated from spicules that are extensively treated with pro-teinase K subsequent to washing with bleach and prior todemineralization appear identical to radiolabeled proteins iso-lated from untreated spicules (data not shown).Identification of SM50 and SM30 Spicule Matrix Pro-

teins—To help determine which spicule matrix proteins wereencoded by the previously cloned SM50 and SM30 genes, an-tisera were raised against the proteins encoded by the clonedcDNAs. While an antiserum raised against SM50 had beenpreviously generated by Richardson et al. (29), it was no longeravailable for these studies. We therefore chose to synthesizeimmunogens for raising antiserum against both of the proteinsencoded by the SM30 and SM50 cDNAs. Maltose-binding pro-tein fusion proteins were constructed using the cDNAs encod-ing SM50 and SM30. The resulting fusion proteins were thenused to immunize rabbits to generate specific antisera.Fig. 4A is a Western blot of a one-dimensional SDS-PAGE

separating untreated spicule matrix protein and endoglycosi-dase F/N-glycosidase F-treated spicule matrix protein. Thisblot was reacted with the anti-SM50 antiserum, and there is aprominent band of about 48 kDa that is detected with thisantiserum. This apparent molecular mass is close to the de-duced molecular mass of the mature processed protein encodedby the cloned SM50 cDNA (21). This observation is the sameresult that Richardson et al. (29) reported. Western blots usingthe preimmune serum resulted in no staining (data not shown).It is also apparent from the results in Fig. 4A that the molec-ular mass does not shift after treatment with endoglycosidaseF/N-glycosidase F, indicating that SM50 is not N-glycosylated.Since there is no consensus N-glycosylation site in the deducedSM50 amino acid sequence, this result is expected. There is anadditional band visible, other than SM50, in each lane at about69 kDa in Fig. 4A. This band is not present in other blots usingthis same antiserum with different spicule matrix preps (e.g.Figs. 6B and 7B). Also, given the apparent molecular mass ofthis band, we postulate that this antiserum is reacting withhuman keratin, a cross-reaction often seen with rabbit poly-clonal antisera.Fig. 4B is a one-dimensional Western blot using the anti-

SM30 antiserum. Akasaka et al. (23) reported that there arebetween 2 and 4 copies of SM30 genes per haploid genome andthat at least two different SM30 genes, designated SM30-a andSM30-b, are arranged tandemly in the genome. It was there-fore not surprising that a doublet with approximate apparentmolecular masses of 43 and 46 kDa reacts with this anti-SM30antiserum. It also appears that the molecular mass of eachdoublet member decreases approximately 3–4 kDa after treat-ment with endoglycosidase F/N-glycosidase F indicating thatthe two SM30 proteins are N-glycosylated. This glycosidasetreatment result is also expected since the deduced amino acidsequence of the SM30-a gene and the SM30 pNG7 cDNA each

contain one consensus N-glycosylation site (22, 23). Westernblotting using the preimmune serum for the anti-SM30 anti-serum resulted in no immunostaining at all (data not shown).The apparent molecular mass of these anti-SM30 reactive

proteins, however, is significantly higher than was expectedbased on the molecular mass of 30.6 kDa from the SM30 cDNApNG7 deduced amino acid sequence (22). The difference in theobserved apparent molecular mass and the derived molecularmass cannot be explained solely by N-glycosylation since theremoval of N-linked carbohydrate moieties only shifts the mo-lecular mass of the SM30 doublet 3–4 kDa. To determine ifO-glycosylation of the SM30 proteins is occurring and contrib-uting to the larger than expected molecular mass, we treatedspicule matrix proteins under alkali condition at 37 °C over-night. These conditions should remove any O-glycosylations(33, 34). The third lane of the Western blot in Fig. 4B showsanti-SM30 antiserum reacted with alkaline-treated spicule ma-trix proteins. There is no visible alteration of the apparentmolecular mass of the SM30 proteins. These results indicatethat these anti-SM30 reactive proteins are not O-glycosylated.Since glycosylation is probably not the reason for the anom-

alous apparent molecular weight of the SM30 proteins, wedevised an experiment to help us determine whether: 1) the

FIG. 4. One-dimensional Western blots of spicule matrix pro-teins using the anti-SM50 and anti-SM30 antisera. A, 0.5 mg ofspicule matrix protein and 0.5 mg of endoglycosidase F/N-glycosidaseF-treated spicule matrix protein were separated on a 10% SDS-PAGEgel. The gel was then subjected to Western blotting and reacted with theanti-SM50 antiserum. B, 0.5 mg of spicule matrix protein, 0.5 mg ofendoglycosidase F/N-glycosidase F-treated spicule matrix protein, and0.5 mg of alkali-treated spicule matrix protein were separated on a 10%SDS-PAGE gel. The gel was then subjected to Western blotting and wasreacted with the anti-SM30 antiserum. The blots in A and B both usedalkaline phosphatase-conjugated secondary antibody and BCIP/NBT assubstrate to visualize the immunoreactive proteins.

FIG. 3. Diagram of the S. purpura-tus spicule matrix proteins. A diagramwas drawn summarizing the distributionof the various spicule matrix proteins sep-arated in gels presented in Fig. 2, A-C.Asterisk (*) indicates strongly radiola-beled spicule matrix protein; I, II, and IIIlabels indicate the three spicule matrixproteins that were used to align the pro-teins that were common among the gels inFig. 2, A-C. The identity SM30-A,SM30-B, and SM50 spicule matrix pro-teins are also designated. NEPHGE,non-equilibrium pH gradient gelelectrophoresis.

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SM30 proteins are migrating with a larger apparent molecularmass because of the inherent chemistry of these proteins, 2) theSM30 proteins are being modified further by post-translationalmodifications other than glycosylation, or 3) the antibody isreacting to other larger spicule matrix proteins that shareepitopes with the protein encoded by the SM30 cDNA. Toaddress these issues, capped RNA transcripts were synthesizedin vitro using the pNG7 cDNA as template. The pNG7 cDNAencodes a full-length SM30 protein (22). This SM30 RNA and[35S]methionine were then co-microinjected into Xenopus oo-cytes. Radiolabeled proteins secreted by SM30 RNA injectedand control oocytes were collected, and the secreted proteinswere then subjected to immunoprecipitation using the anti-SM30 antiserum. The immunoprecipitates were then sepa-rated by SDS-PAGE. The results of this experiment are shownin Fig. 5A. From this gel, it is apparent that a 46-kDa se-creted protein is synthesized by the oocytes injected withpNG7 SM30 mRNA. Fig. 5A also shows that the SM30 pro-tein synthesized by the Xenopus oocyte decreases in apparentmolecular mass by 3–4 kDa when it is treated with endogly-cosidase F/N-glycosidase F.SM30 RNA synthesized from the pNG7 cDNA was also

translated in vitro using rabbit reticulocyte lysate, which doesnot post-translationally modify translation products. Fig. 5Bshows that the reticulocyte lysate synthesizes an approxi-mately 32-kDa product which is the molecular masss of thededuced amino acid sequence of the pNG7 SM30 cDNA that

contains its signal sequence. Given these results, it appearsthat one or more post-translation modifications other thanN-glycosylation produces the anomalous electrophoretic migra-tion of the SM30 proteins. Since the Xenopus oocyte seems to bemodifying the pNG7 SM30 protein correctly, a convenient invitro assay exists to determine the nature of the post-transla-tional modifications occurring to SM30 proteins. We are pres-ently attempting to identify the as of yet unknown post-trans-lational modification(s) of the SM30 proteins with this system.To further determine if the observed SM30 doublet was real

or apparent, we also ran gels with spicule matrix proteinssubjected to higher amounts of reducing agent and higher SDSconcentrations. However, these treatments did not alter themigration or presence of the anti-SM30 reactive doublet (datanot shown). Since the 43- and 46-kDa anti-SM30 reactive pro-teins appear to encode genuine SM30 proteins, these proteinsare designated SM30-A and SM30-B, respectively.To determine which of the spots on the two-dimensional gels

of the spicule matrix proteins are actually the SM30 and SM50proteins, Western blots of two-dimensional gels were done. Fig.6A is a two-dimensional Western blot using the anti-SM30antiserum. The first dimension of this gel was an equilibriumisoelectric focusing gel using ampholytes with a range of 2.5–6.5 (similar to the first dimension of the gel in Fig. 2B). Fromthis blot it is apparent that the anti-SM30 antiserum reactswith two closely migrating spots at the acidic end of the gelwith the same apparent molecular weight as the SM30-A andSM30-B proteins in Fig. 4B. When this gel is compared to a gelseparating labeled spicule matrix proteins that was run inparallel, it is apparent that SM30-A and SM30-B are two of themore acidic and highly radiolabeled spicule matrix proteins.The identity of the SM30-A and SM30-B proteins are marked inFig. 3.Fig. 6B is a two-dimensional Western blot using the anti-

SM50 antiserum. The first dimension of this blot was a non-equilibrium pH gradient gel using ampholytes with a pH rangeof 3–10 and the gel run in the basic direction (similar to thefirst dimension of the gel in Fig. 2C). From this blot it isapparent that the anti-SM50 antiserum reacts with a singleprotein that migrates with an apparent molecular mass of 48kDa at the most alkaline portion of the gel. When compared toa gel separating labeled spicule matrix protein that was run inparallel, it is apparent that SM50 is the spicule matrix proteinwith the most alkaline isoelectric point. This observation isconsistent with the pI of the deduced amino acid sequence ofthe SM50 cDNA being approximately 12 (21). The identity ofthe SM50 protein is marked in Fig. 3.Comparison of Spicule Matrix Proteins with the Integral

Matrix Proteins of Adult Spines—The spines of adult sea ur-chins have some of the same interesting physical properties asthe embryonic spicules such as an aligned crystal axis andgreater fexural strength (1, 4–6, 42). It is also known fromNorthern blot analysis that RNAs complementary to the pre-viously cloned SM50 and SM30 cDNAs are expressed in adultspines (22, 29). Therefore, we thought that comparing the em-bryonic spicule matrix proteins with the integral matrix pro-teins of the adult sea urchin spines would be instructive. Weused the antibodies we have generated here to examine theintegral matrix proteins of the adult spines.Comparison of one-dimensional Western blots of embryonic

spicule matrix protein and adult spine integral matrix protein,using an antiserum raised against all of the sea urchin embry-onic spicule matrix proteins, is shown in Fig. 7A. This figurereveals extensive cross-reactivity between the two tissues.Qualitatively, the most immunoreactive proteins in adult spinematrix seem to have apparent molecular masses larger than

FIG. 5. In vitro translation of SM30 RNA by Xenopus oocytesand reticulocyte lysate. SM30 RNA was synthesized in vitro usingthe SM30 cDNA clone pNG7 (22) as the template. A, Xenopus oocyteswere microinjected with in vitro synthesized SM30 RNA and [35S]me-thionine. The supernatant of the SM30 RNA injected oocytes whichcontained radiolabeled, secreted proteins were collected 2 days later(control). A portion of this supernantant was treated with endoglycosi-dase F/N-glycosidase F (glycosidase treated). The untreated and glyco-sidase-treated supernatants were then subjected to immunoprecipita-tion using the preimmune and anti-SM30 antisera. The resultingprecipitates were separated on a 10% SDS-PAGE gel, processed forfluorography, and exposed to x-ray film. The anti-SM30 antiserumspecifically immunoprecipitates a broad 46-kDa band from the un-treated oocyte supernantant (indicated as untreated SM30). This banddecreases in apparent molecular mass approximately 3–4 kDa whenthe oocyte supernantant is glycosidase treated (indicated as treatedSM30). B, rabbit reticulocyte lysate was used to translate in vitro SM30RNA, control RNA (Xenopus elongation factor 1), and no added RNA inthe presence of [35S]methionine. Equal portions of the lysates thatcontained the various radiolabeled protein products were then sepa-rated on a 10% SDS-PAGE gel and processed for fluorography. Theprotein product synthesized using the SM30 RNA has an approximately32-kDa apparent molecular mass.

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those from the spicule matrix. One exception is a band atapproximately 120 kDa which has a similar sized counterpartin the spicule matrix protein. We know, however, that theembryonic spicule matrix band at 120 kDa is composed of a fewdifferent proteins of that same size in the spicule matrix (seeFigs. 2 and 3).A Western blot using anti-SM50 antiserum (Fig. 7B) reveals

that SM50 has the same apparent molecular mass in spinematrix as it does in spicule matrix, an observation seen byRichardson et al. (29) using a different anti-SM50 antiserum.One-dimensional blot analysis of spicule and spine matrix pro-teins using the anti-SM30 antiserum reveals differences be-tween these two tissues. This result is shown in Figs. 7, C andD. While anti-SM30 reacts with spicule matrix bands withapparent molecular masses of 43 and 46 kDa, this antiserumreacts with a doublet of 46 and 49 kDa in spine matrix (see Fig.7C). These two sets of SM30 doublets in Fig. 7C are located atexactly the same apparent molecular mass as two prominentimmunoreactive doublets in the spicule and spine lanes in Fig.7A that reacted with the anti-total spicule matrix antiserum.To determine whether the apparent size difference in the dou-blets that react with anti-SM30 is caused by differential glyco-sylation, glycosidase-treated spicule and spine matrix proteinwere analyzed by Western blotting. As can be seen in Fig. 7D,both spicule and spine doublets have reduced apparent molec-ular masses of about 3 kDa each after glycosidase treatment.However, a size difference between the SM30 doublets presentin spicule and spine matrix persists. This finding suggests thatthe difference in apparent molecular weight is not because ofdifferential N-glycosylation. Rather, it suggests that the 49-kDa protein is a different form of SM30 and/or it is post-translationally modified (other than N-glycosylation) differ-ently than SM30-A or SM30-B. We designate this apparentadult isoform of SM30 as SM30-C. The results in Fig. 7, C andD, also suggest that the 43-kDa SM30-A protein is an embry-onic isoform of SM30 since there is no protein of that size in theadult spine matrix lane. The 46-kDa protein, SM30-B, is ap-parently expressed in both spicules and adult spines. Of course,at this point, we cannot rule out the possibility that the 46-kDaproteins in the embryonic spicule matrix and the adult spinematrix are encoded by different genes. Akasaka et al. (23) haveshown that there are up to four different SM30 genes. But, atthis time, we have no reason to invoke that possibility.

FIG. 7. Comparison of embryonic spicule matrix proteins andadult spine integral matrix proteins. A, embryonic spicule matrixprotein and adult spine matrix protein were separated on a 10% SDS-PAGE gel and subjected to Western blotting analysis using a polyclonalantiserum raised against all of the spicule matrix proteins. B, embry-onic spicule matrix protein and adult spine matrix protein were sepa-rated on a 10% SDS-PAGE gel and subjected to Western blottinganalysis using the anti-SM50 antiserum. C, embryonic spicule matrixprotein and adult spine matrix protein were separated on a 10% SDS-PAGE gel and subjected to Western blotting analysis using the anti-SM30 antiserum. D, endoglycosidase F/N-glycosidase F-treated embry-onic spicule matrix protein and adult spine matrix protein wereseparated on a 10% SDS-PAGE gel and subjected to Western blottinganalysis using the anti-SM30 antiserum. Alkaline phosphatase-conju-gated secondary antibody and BCIP/NBT as substrate were used tovisualize the immunoreactive proteins in these four blots.

FIG. 6. Two-dimensional Western blots of spicule matrix proteins using the anti-SM30 and anti-SM50 antiserum. A, 0.5 mg ofunlabeled spicule matrix protein was separated in the first dimension on an isoelectric focusing gel using ampholytes with a pH range of 2.5 to 6.5as described under “Experimental Procedures.” The second dimension was a 10% SDS-PAGE gel. The proteins were subjected to Western blottingusing the anti-SM30 antiserum. B, 0.5 mg of unlabeled spicule matrix protein was separated in the first dimension on a nonequilibrium pH gradientgel run in the basic direction. The pH range of ampholytes used was 3 to 10. The second dimension was a 10% SDS-PAGE gel. The proteins weresubjected to Western blotting analysis using the anti-SM50 antiserum. The immunoreactive proteins in these two blots were visualized usingchemiluminescence. NEPHGE, non-equilibrium pH gradient gel electrophoresis.

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Comparison of the Sequence of the SM30 Proteins with C-typeLectin Family of Proteins—Harkey et al., (27) found that alarge portion of the PM27 and SM50 proteins have some sim-ilarity to the CRD of the C-type lectin family of proteins; wetherefore decided to determine if this similarity exists for theSM30 proteins. A search of the Swiss-Prot protein data base(release 31) using the BLITZ automatic electronic mail serverfor the MPsrch search program (version 1.5) revealed a signif-icant similarity (less than e25 probability for randomness) be-tween the SM30 protein encoded by the SM30-a gene and anumber of C-type lectin proteins. The 20 proteins most similarto SM30 were all C-type lectins. Fig. 8 illustrates the similaritybetween the SM30 protein encoded by SM30-a and the 10 mostsimilar proteins. It is apparent that residues 80 through 210 ofthe SM30-a protein are fairly similar to the CRD of C-typelectins listed in Fig. 8 suggesting that SM30-a protein has aC-type lectin CRD. The percentage of identical amino acids inthe C-type lectins over the portion aligned with SM30-a rangefrom 20.7 to 28.8%. The percentage of identical or conservedamino acid substitutions range from 31.5 to 44.9%. A high level

of similarity occurs at the amino acid residues that make up thelarge and small hydrophobic core domains of typical C-typelectins that were described by Weis et al. (44). There are twodifferences between SM30 and C-type lectins particularlyworth noting. 1) SM30-a protein lacks a pair of cysteine resi-dues that are conserved in other C-type lectins and that areinternal to the two cysteine residues that are conserved inSM30-a protein and 2) there is imperfect matching of SM30-aprotein with the known conserved residues making up the twocalcium ion binding sites that are typically found in C-typelectin CRD.When SM30-a was initially cloned and characterized, it was

noted there were nine amino acid differences between the pro-tein encoded by it and the protein encoded by the SM30 pNG7cDNA (23). It was unclear if these differences reflected the wellknow polymorphisms of sea urchin genomic sequences or if itreflected that SM30-a and pNG7 encode different SM30 pro-teins. It is interesting to note that seven of the nine amino aciddifferences between the SM30-a protein and the pNG7 SM30protein occur over the approximately 40% portion of the SM30

FIG. 8. The similarity between SM30-a protein and proteins containing a C-type lectin carbohydrate recognition domain. A regionof SM30-a protein is aligned with various proteins containing a C-type lectin carbohydrate recognition domain. Amino acids that are boxed in blackindicate identity with the SM30-a protein. Amino acids that are boxed in gray indicate a conservative amino acid substitution.Numbers in bracketsnext to protein names indicate the residue numbers of the various proteins that are aligned in the figure. Dash (-) indicates gaps that were addedto help align the sequences. Numbers at the top of the alignment refer to amino acid residue number for the SM30-a protein (accession numberP28163). Protein names are in Swiss Prot format. LITH RAT, rat lithostathine precursor (accession number P10758); LITH HUMAN, humanlithostathine precursor (accession number P05451); MANR HUMAN, human macrophage mannose receptor (accession number P22897);PAP1 HUMAN, human pancreatitis-associated protein 1 precursor (accession number Q06141); LITH BOVIN, bovine lithostathine precursor(accession number P23132); PAP1 RAT, rat pancreatitis-associated protein 1 precursor (accession number P25031); PAP1 MOUSE, mousepancreatitis-associated protein 1 precursor (accession number P35230); TETN HUMAN, human tetranectin precursor (accession numberP05452); LECA PLEWA, Pleuradeles waltii lectin precursor (accession number Q02988); LECE ANTCR, Anthocidaris crassispina echinoidin(accession number P06027). Arrows above the SM30-a protein sequence indicate residues that are different in the protein encoded by pNG7 SM30cDNA. The different amino acid residues in the pNG7 protein are indicated on top of the arrows. Symbols for the conserved structure and aminoacid residues of C-type lectins determined by Weis et al. (44): u 5 aliphatic; f 5 aromatic; x 5 aliphatic or aromatic; Z 5 E or Q; B 5 D or N; W5 side chain containing oxygen (D, N, or Q); W, D, P, and C 5 single letter symbols of amino acids; 1, first Ca21 binding site; 2, second Ca21 site;S, small hydrophobic core; L, large hydrophobic core.

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proteins that is similar to the CRD of C-type lectins. Arrows inFig. 8 indicate where these differences occur. Most of thesedifferences alter residues shown in the present study to beconserved between SM30-a protein and C-type lectins or inresidues shown by Weis et al. (44) to be conserved amongC-type lectins. While it is premature to conclude too much fromthese differences, these findings suggest that SM30-a andpNG7 SM30 may be different forms of the SM30 protein.

DISCUSSION

Acidic integral matrix proteins isolated from calcitic adultsea urchin exoskeletal tissues have been shown to bind tospecific faces of calcite crystal in vitro (4). It has also beenshown that the intercalation of the matrix proteins within thecalcite is responsible for some of the physical properties of adultsea urchin skeletal elements (5, 6). However, these studies didnot characterize the integral matrix proteins other than deter-mining partial amino acid composition. There is evidence thatthe integral matrix proteins of adult mineralized tissues andthe integral matrix proteins of embryonic spicules control sim-ilar physical properties of these calcitic tissues (6). In addition,transcripts complementary to the previously cloned spiculematrix SM50 and SM30 cDNAs are expressed in adult miner-alized tissues (22, 29). Therefore we decided to further charac-terize the embryonic spicule matrix proteins and compare themto adult integral matrix proteins.Our studies presented here show that there are many more

spicule matrix proteins than were detected in previous studies.A dozen prominently radiolabeled spicule matrix proteins andsome three dozen less prominently radiolabeled spicule matrixproteins are detected. The apparent molecular mass of theproteins range from 20 kDa to over 100 kDa. Most of theseproteins, including the more prominent ones, are acidic. Wealso show that there are several less intensely radiolabeledspicule matrix proteins that have more alkaline isoelectricpoints. The predicted chemistry of the spicule matrix proteinsis consistent with that found for other integral matrix proteinsof calcified structures for other organisms (8, 9). The resultspresented in this article further indicate that the adult integralmatrix proteins are similar to embryonic spicule matrix pro-teins since there is significant Western blot cross-reactivitywith the anti-total spicule matrix antiserum. However, sincethe one-dimensional SDS-PAGE Western antibody stainingpattern is different from spicule matrix protein’s pattern, thissuggests that there are several different integral matrix pro-teins, or at least several different forms of integral matrixprotein, in the adult spine.Western blot analysis of the previously cloned SM50 gene

shows that the SM50 protein is the spicule matrix protein withthe most alkaline isoelectric point and that it has an apparentmolecular mass of approximately 48 kDa. This result is con-sistent with the observations of Livingston et al. (28) andRichardson et al. (29). Livingston et al. (28) cloned the L. pictushomologue of SM50. This 34-kDa protein, designated LSM34,is also not glycosylated and has an alkaline pI as determinedfrom its derived amino acid sequence. The glycosidase resultsof the present paper and Livingston et al. (28) are to be ex-pected since there is no N-linked glycosylation consensus se-quence in the derived amino acid sequence of the S. purpuratusSM50 cDNA (21) and the L. pictus LSM34 cDNA (28). Richard-son et al. (29) generated an anti-SM50 specific polyclonal anti-serum and they observed that it reacted with a single band ofapproximately 48 kDa in S. purpuratus embryonic spicules andadult spines.It is particularly interesting that the Western blotting anal-

ysis using the anti-SM30 antiserum reveals that there aremultiple forms of the SM30 protein in the sea urchin embryonic

spicule matrix and in the adult spine matrix. This result isconsistent with our assertion that adult spine integral matrixcontains some proteins that are different from embryonic spi-cule matrix. When the SM30 cDNA was originally isolated,Northern blots revealed only one band hybridizing with theSM30 cDNA probe (22). However, recent work (23) has demon-strated that SM30 is a small multigene family with betweentwo and four copies of the SM30 gene present per haploidgenome of S. purpuratus. A genomic clone was also isolated andit was found that at least two SM30 genes are tandemly ar-ranged (designated SM30-a and SM30-b). Therefore, it is con-sistent, given these findings, to see a doublet of SM30 proteinsin the spicule matrix of 43 and 46 kDa (designated as SM30-Aand SM30-B, respectively) and a doublet of SM30 protein inadult spine matrix of 46 and 49 kDa (designated SM30-B andSM30-C, respectively).Harkey et al. (27) reported that SM50 and the PM27 proteins

are similar to C-type lectin proteins and we now report thatSM30 is also similar to the CRD domain of C-type lectins. TheC-type lectins are a very heterogenous family of proteins thatare most often involved in recognition events outside cells (seefor reviews, Refs. 45–47). Usually these recognition events aremediated through a Ca21 dependent binding to carbohydratemoieties. However, certain C-type lectins have also been shownto bind proteins directly (48), and to act as antifreeze moleculesin coldwater fish (49).It is interesting to note that two types of C-type lectins that

were found to be most similar to SM30-a protein in our studieshere have previously been found to be involved in the formationof mineralized structures within animals. One of these C-typelectins is tetranectin which is a blood and extracellular matrixcomponent. Wewer et al. (50) reported localization of tetranec-tin in mineralizing mouse bone osteoblastic cells that weredifferentiating in vitro. However, the precise molecular roletetranectin plays in osteogenesis remains unknown. The otherC-type lectin previously found to be involved in mineralizedstructures is called lithostathine (formerly called pancreaticstone protein (51)). Lithostathine is present in pancreatic juicesand it is believed to bind calcium carbonate and prevent cal-cium carbonate from precipitating and forming calcitic pancre-atic stones (see for review, Ref. 52). However, the 11 amino acidpeptide sequence at the amino end of lithostathine that isthought to interact with calcium carbonate is not within theCRD of lithostathine and it is not well conserved in SM30 orSM50 (data not shown). So whether there is homology of func-tion of SM30-a protein and lithostathine to go along with thesimilarity of their sequences remains enigmatic. These find-ings, however, raise several interesting questions. Could therebe other regions of these spicule matrix proteins that bindcalcium carbonate? Could the similarity to C-type lectins bereflective of the SM30 and SM50 proteins binding the carbo-hydrate moieties of other spicule matrix proteins? SM30-a pro-tein has imperfect matching at the calcium ion binding sitestypically found in C-type lectins. Does SM30-a protein bindCa21? Many attempts in our laboratory over the years bydifferent techniques have failed to reveal Ca21 binding toSM30. Why are so many of the known proteins which areclosely associated with spicule formation similar to C-type lec-tins? Experiments are underway to address these questions.It remains undetermined which of the SM30 proteins iden-

tified in the present paper are encoded by the SM30-a, SM30-b,or the pNG7 SM30 cDNA. Results presented in this paper pointout that 7 out of 9 differences in the amino acid sequencebetween the protein encoded by the pNG7 SM30 cDNA and theprotein encoded by SM30-a occur over the approximately 40%portion of the SM30 proteins that comprise the region similar

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to the CRD of C-type lectins. Most of these differences alterconserved residues suggesting that they may encode for differ-ent forms of the SM30 proteins. In addition to the studiespresented here, unpublished RNase protection studies showthat SM30-a gene transcript is expressed in the sea urchinembryo and not in the adult spine.2 Since SM30-A proteinseems to be present in embryonic spicule and not in adultspines, this unpublished finding suggests that SM30-A may beencoded by the SM30-a genomic sequence. The complete se-quence of SM30-b is not yet known (23) and results presentedin the current paper suggest that there is at least a third SM30gene that has not been cloned. Therefore, until the completesequence of all of the SM30 genomic sequences as well as theirexpression patterns are known, we cannot be sure if SM30-aencodes SM30-A or if pNG7 SM30 cDNA encodes for a SM30protein different from the one encoded by SM30-a. Studiesaddressing these issues are underway.Our enumeration, characterization, and comparisons of

spine and spicule matrix proteins complement studies of Ber-man et al. (4–6). They observed that sea urchin integral matrixproteins are not as acidic as some other invertebrate integralmatrix proteins. We have found that there is a range of acidicto alkaline pI values for the spicule matrix proteins, althoughthe net pI of the proteins as a whole is acidic (16). Berman et al.(4) showed that the integral matrix proteins from adult seaurchin mineralized tissues, but not the more acidic integralproteins from the mollusc Mytilus californus prismatic layer,were able to bind specific calcite crystal faces, even though themollusc proteins are known nucleators of calcite when they areadsorbed on a rigid substrate. Berman et al. (6) also studieddifferences in texture of calcite crystals among calcitic tissuesfrom different taxonomic groups, including analysis of sea ur-chin embryo spicules and adult spines. They showed that themanipulation of crystal structure is under biological controland that the integral matrix proteins probably play a role inthis control. The studies presented here provide a fuller char-acterization of the embryo spicule and adult spine matrix pro-teins integral matrix proteins. Future studies looking at phys-ical interactions of individual spicule matrix proteins withcalcite crystals in vitro will be particularly informative.

Acknowledgments—We acknowledge Patricia Hamilton and AdinaBailey for their excellent technical assistance as well as Steve Benson,William Lennarz, Martin Brown, and many members of the Wilt labo-ratory for their helpful discussions and suggestions during the course ofthese studies. We gratefully acknowledge Michael Wu of the Gerhardtlaboratory for providing and microinjecting the Xenopus oocytes, andRichard Kostriken for advice on the construction and expression ofbacterial fusion proteins. We also gratefully acknowledge Richard Ko-striken, Eric Ingersoll, Brian Livingston, and Carole Ungvarsky fortheir critical reading of the manuscript.

REFERENCES

1. Emlet, R. B. (1982) Biol. Bull. 163, 264–2752. Okazaki, K., and Inoue, S. (1976) Dev. Growth and Differ. 18, 413–4343. Okazaki, K., McDonald, K., and Inoue, S. (1980) in The Mechanisms of Bi-

omineralization in Animals and Plants (Omori, M., andWatabe, N., eds) pp.159–168, Tokai University Press, Tokyo

4. Berman, A., Addadi, L., and Weiner, S. (1988) Nature 331, 546–5485. Berman, A., Addadi, L., Kvick, Å., Leiserowitz, L., Nelson, M., and Weiner, S.

(1990) Science 250, 664–6676. Berman, A., Hanson, J., Leiserowitz, L., Koetzle, T., Weiner, S., and Addadi, L.

(1993) Science 259, 776–7797. Aizenberg, J., Hanson, J., Ilan, M., Leiserowitz, L., Koetzle, T. F., Addadi, L.,

and Weiner, S. (1995) Faseb J. 9, 262–2688. Weiner, S. (1984) Am. Zool. 24, 945–9519. Weiner, S. (1986) CRC Crit. Rev. Biochem. 20, 365–40810. Okazaki, K. (1975) Am. Zool. 15, 567–58111. Decker, G., and Lennarz, W. J. (1988) Development 103, 231–24712. Ettensohn, C. A. (1991) in Cell-Cell Interactions in Early Development, 49th

Symposium of the Society for Developmental Biology (Gerhart, J., ed) pp.175–201, Wiley-Liss, Inc., New York

13. Benson, S. C., and Wilt, F. H. (1992) in Calcification in Biological Systems(Bonucci, E., ed) pp. 157–178, CRC Press, Ann Arbor, MI

14. Wilt, F. H., Killian, C. E., and Livingston, B. T. (1993) in Chemistry andBiology of Mineralized Tissues (Slavkin, H., and Price, P., eds) pp. 85–92,Excerpta Medica, New York

15. Benson, S. C., Jones, E. M., Benson, N. C., and Wilt, F. (1983) Exp. Cell Res.148, 249–253

16. Benson, S. C., Benson, N. C., and Wilt, F. H. (1986) J. Cell Biol. 102,1878–1886

17. Venkatesan, M., and Simpson, R. T. (1986) Exp. Cell Res. 166, 259–26418. Sucov, H. M., Benson, S., Robinson, J. J., Britten, R. J., Wilt, F., and Davidson,

E. H. (1987) Dev. Biol. 120, 507–51919. Benson, S., Sucov, H., Stephens, L., Davidson, E., and Wilt, F. (1987)Dev. Biol.

120, 499–50620. Killian, C. E., and Wilt, F. H. (1989) Dev. Biol. 133, 148–15621. Katoh-Fukui, Y., Noce, T., Ueda, T., Fujiwara, Y., Hashimoto, N., Higashi-

nakagawa, T., Killian, C., Livingston, B., Wilt, F., Benson, S., Sucov, H.,and Davidson, E. H. (1991) Dev. Biol. 145, 201–202

22. George, N. C., Killian, C. E., and Wilt, F. H. (1991) Dev. Biol. 147, 334–34223. Akasaka, K., Frudakis, T. N., Killian, C. E., George, N. C., Yamasu, K.,

Khaner, O., and Wilt, F. (1994) J. Biol. Chem. 269, 20592–2059824. Sucov, H. M., Hough-Evans, B. R., Franks, R. R., Britten, R. J., and Davidson,

E. H. (1988) Genes and Develop. 2, 1238–125025. Makabe, K. W., Kirchhamer, C. V., Britten, R. J., and Davidson, E. H. (1995)

Development 121, 1957–197026. Frudakis, T. N., and Wilt, F. H. (1995) Dev. Biol. 172, 230–24127. Harkey, M. A., Klueg, K., Sheppard, P., and Raff, R. A. (1995) Dev. Biol. 168,

549–56628. Livingston, B. T., Shaw, R., Bailey, A., and Wilt, F. (1991) Dev. Biol. 148,

473–48029. Richardson, W., Kitajima, T., Wilt, F., and Benson, S. (1989) Dev. Biol. 132,

266–26930. Benson, S., Smith, L., Wilt, F., and Shaw, R. (1990) Exp. Cell Res. 188, 141–14631. Meier, T., Arni, S., Malarkannan, S., Poincelet, M., and Hoessli, D. (1992)

Anal. Biochem. 204, 220–22632. Nesbitt, S., and Horton, M. A. (1992) Anal. Biochem. 206, 267–27233. Florman, H. M., and Wassarman, P. M. (1985) Cell 41, 313–32434. Sharon, N. (1975) Complex Carbohydrates, Their Chemistry, Biosynthesis, and

Function, pp. 73–78, Addison-Wesley Publishing Co., Reading, MA35. O’Farrell, P. H. (1975) J. Biol. Chem. 250, 4007–402136. Laemmli, U. K. (1970) Nature 227, 680–68537. Dreyfus, G., Adams, S. A., and Choi, Y. D. (1984) Mol. Cell. Biol. 4, 415–42338. Laskey, R. A., and Mills, A. D. (1975) Eur. J. Biochem. 56, 335–34139. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring

Harbor Laboratory, Cold Sping Harbor, NY40. Towbin, H., Stachelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A.

76, 4350–435441. Dunbar, B. S., Kimura, H., and Timmons, T. M. (1990)Methods Enzymol. 182,

441–45942. Raup, D. M. (1966) in Physiology of Echinodermata (Boolootian, R. A., ed) pp.

379–395, Intersciences Publishers, London43. Harkey, M. A., and Whiteley, A. H. (1983) Dev. Biol. 100, 12–2844. Weis, W. I., Kahn, R., Fourme, R., Drickamer, K., and Hendrickson, W. A.

(1991) Science 254, 1608–161545. Drickamer, K. (1993) Prog. Nucleic Acids Res. Mol. Biol. 45, 207–23246. Drickamer, K. (1993) Curr. Opin. Struct. Biol. 3, 393–40047. Drickamer, K., and Taylor, M. E. (1993) Ann. Rev. Cell Biol. 9, 237–26448. Bettler, B., Texido, G., Raggini, S., Ruegg, D., and Hofstetter, H. (1992) J. Biol.

Chem. 267, 185–19149. Ng, N. F. L., and Hew, C. L. (1992) J. Biol. Chem. 267, 16069–1607550. Wewer, U. M., Ibaraki, K., Schjørring, P., Durkin, M. E., Young, M. F., and

Albrechtson, R. (1994) J. Cell Biol. 127, 1767–177551. Sarles, H., Dagorn, J. C., Giorgi, D., and Bernard, J. P. (1990) Gastroentroen-

terology 99, 900–90152. Dagorn, J. C. (1993) in The Pancreas (Go, V. L. W., DiMagno, E. P., Gardner,

J. D., Lebenthal, E., Reber, H. A., and Scheele, G. A., eds) pp. 253–263,Raven Press, Ltd., New York2 C. E. Killian and F. H. Wilt, unpublished data.

Sea Urchin Spicule Matrix Proteins 9159

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Christopher E. Killian and Fred H. Wilt Embryonic Spicules purpuratus

StrongylocentrotusCharacterization of the Proteins Comprising the Integral Matrix of

doi: 10.1074/jbc.271.15.91501996, 271:9150-9159.J. Biol. Chem. 

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