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Characterization of a cDNA encoding a protein involved in formation of the skeleton during development of the sea urchin Lytechinus pictus
DEVELOPMENTALBIOLOGY
148,47%48u
(19%)
Characterization of a cDNA Encoding a Protein Involved in Formation of the Skeleton during Development of the Sea Urchin Lytechinus pictus BRIAN Department
of Molecular
T. LIVINGSTON,’ and
Cell
Biology,
ROBIN SHAW, 5’7.9 La&
Sciences
ADINA
Addition,
BAILEY, Uniwrsity
AND FRED WILT of Culifbrnia,
Berkeley,
Cula&Gz
9~720
In order to investigate the role of proteins in the formation of mineralized tissues during development, we have isolated a cDNA that encodes a protein that is a component of the organic matrix of the skeletal spicule of the sea urchin, Lytechinus fictus. The expression of the RNA encoding this protein is regulated over development and is localized to the descendents of the micromere lineage. Comparison of the sequence of this cDNA to homologous cDNAs from other species of urchin reveal that the protein is basic and contains three conserved structural motifs: a signal peptide. a proline-rich region, and an unusual region composed of a series of direct repeats. Studies on the protein encoded by this cDNA confirm the predicted reading frame deduced from the nucleotide sequence and show that the protein is secreted and not glycosylated. Comparison of the amino acid sequence to databases reveal that the repeat domain is similar to cc>19Y1 Academic Press, Inc. proteins that form a unique B-spiral supersecondary structure. INTRODIJCTION
The role of proteins that participate in the formation of mineralized tissue is poorly understood, largely due to the complex nature of the tissues studied. Isolation and characterization of the protein components of mineralized structures are important first steps in elucidating their function. The endoskeletal spicule of sea urchin larvae has provided a simple system to begin the study of individual proteins involved in the formation of mineralized tissue during development. The spicule is formed during embryonic development by a group of cells, called micromeres, which arise at the vegetal pole of the embryo during the fourth cleavage division. These cells give rise to the primary mesenchyme cells, which migrate into the blastocoel cavity and in turn differentiate into the skeletal spicules (Wilt and Benson, 1988). The organic matrix of the spicule has been isolated (Benson et al., 1986), and studies of the matrix have shown that it is composed of a small number of proteins that are predicted to be highly acidic, N-linked glycoproteins. This is in agreement with the nature of organic matrices found in other mineralized tissues (Weiner, 1984). cDNA and genomic clones that encode a spicule matrix protein (SM50) were first isolated from the sea urchin Stmngylocentrotus purpuratus. The amino acid sequence of the protein predicted by the nucleotide sequence of the gene was initially reported to be acidic and to contain sequences involved in secretion and N-linked
’ To whom
correspondence
should
glycosylation (Sucov et o,l, 1987). However, sequence information from homologous cDNAs from two other species, Hemicentrotus’ and Lytechinus picks have revealed an error in the nucleotide sequence of the SM50 gene (Katoh-Fukui et ub, 1991). The new derived amino acid sequence for the SM50 protein and its homologues now predicts them to be basic, nonglycosylated proteins. We report here the cloning and characterization of a cDNA (pLSM1) from Lytech,inus p&us encoding a protein, LSM34, that is the homologue of SM50, the S. purpuratus gene encoding a spicule matrix protein. The characterization of a homologous cDNA from a species that diverged from S purpuratus some 60 million years ago has allowed us to examine the sequence for conserved regions that may encode important functional domains, and to infer how the two genes have evolved from a common ancestor. We have also begun to characterize the protein (LSM34) itself. The 34-kDa protein is secreted, is not glycosylated, and contains several conserved domains, one of which contains an unusual six- to seven-amino acid repeat. MATERIALS
473
METHODS
Culturing Sea Urchin Embryos L. pictus gametes were obtained, eggs were fertilized, and embryos were cultured according to conventional methods (Hinegardner, 1967). Fertilization membranes were removed as described by Hall (1978).
’ T. Higashinakagawa,
be addressed.
AND
personal
communication. OOIZ-1606/91 Copyright All rights
$3.00
c 1991 by Academic Press, Inc. of reproduction in any form reserved.
474
DEVELOPMENTALBIOLOGY V0~~~~148,1991
Isolation
of RNA
Mesenchyme and endoderm cell fractions were isolated from pluteus-stage larvae using a procedure described by Harkey and Whitely (1980). RNA was isolated from these cells using the method of March et al. (1985) as modified by Brandhorst (personal communication) and described by Stephens et al. (1989). Poly(A)+ RNA was isolated on oligo-dT cellulose columns as described by Sambrook et al. (1989). RNA Probe Synthesis
Single stranded RNA probes for SM50, labeled with [32P]UTP, (800 Ci/mmole) were synthesized from the pHS72 plasmid (Benson et al., 1987) using the SP6 promoter of pSP65 (Melton et al., 1984). Probes for pLSM were synthesized using rH]UTP from the T3 promoter of Bluescript (Stratagene), using the cloned cDNA described herein. XgtlO
Library
Construction
and Screening
cDNA was synthesized from 5 pug of poly(A)+ RNA using Pharmacia’s cDNA synthesis system according to the manufacturers instructions. EcoRI linkers were added, the cDNA was ligated into the EcoRI site of XgtlO, and packaged into phage particles using Gigapack Gold (Stratagene) packaging extracts, as described by the manufacturer. Phage were plated and transferred to nitrocellulose using standard procedures (Sambrook et al., 1989). Hybridizations using the SM50 RNA probe were at 60°C in 4~ SET buffer (80 mM Tris (pH 8.0), 0.6 M NaCl, 4 mM EDTA), 5~ Denhardts, and 100 yg yeast RNA/ml for 18 hr. Filters were washed three times at 60°C in 0.3~ SET and positive plaques detected by autoradiography. In Situ Hybridizations In situ hybridizations were carried out as described by Cox et al. (1984) using [3H]UTP-labeled probes. Emulsion coated slides were exposed for 14 days. DNA Sequencing
DNA was sequenced using Sequenase (USB) sequencing system according to standard dideoxy-sequencing methods. Translation in Xenopus Oocytes
Full length, capped, sense strand RNA was synthesized from the pLSM1 cDNA, subcloned into the EcoRI site of pGEM4Z (Promega), using SP6 polymerase, as described by Sambrook et al. (1989). Oocytes were obtained, injected, and cultured as described by Leaf et al.
(1990). [35S]Methionine (40-50 nl, 0.6-0.75 PCi) was injected into oocytes to label newly synthesized proteins. Culture medium was removed after 48 hr, and immunoprecipitations were performed according to Moore and Kelly (1985), using antibody raised against the total spicule matrix of S. purpuratus (Benson et al, 1986). Electrophoresis was in 12.5% acrylamide gels as described by Laemmli (1970) and modified by Dreyfus et al. (1984). Immunoblotting
Electrophoresis was done as described above. Immunoblotting was done as described by Towbin et al. (1979), with modifications described by Johnson et al. (1984). Binding of antibody to proteins on the nitrocellulose was carried out using a chemiluminescent detection system (Amersham) as described by the manufacturer. Gtycosidase Digestions
Glycopeptidase F (Boehringer-Mannheim) digestions were carried out at 37°C for 18 hr, as described by the manufacturer. Electrophoresis was carried out as described above. RESULTS Developmental Expression
The cDNA clone pLSM1 was isolated from a L. pictus XgtlO cDNA library using an S. purpwatus cDNA, pHS72, known to encode a spicule matrix protein, as probe. The timing of the expression of this cDNA was examined using Northern blot analysis. Total RNA from various stages of development was subjected to electrophoresis, blotted to nitrocellulose, and probed with radioactive transcripts from the cDNA. The RNA was first expressed at blastula and continued to be detected through the pluteus stage (data not shown). This is consistent with the pattern of expression seen in S. purpuratus (Benson et al., 1987). The cellular localization of transcripts was examined using in situ hybridization with 3H RNA probes transcribed from the cDNA clone of L. pi&s. Figure 1 shows a representative sample of the results. The transcripts are localized exclusively in the primary mesenchyme cells within the blastocoel. Again this is consistent with the expression of the S. pwpuratus homologue, and suggests the pLSM1 cDNA encodes a spicule matrix protein. Sequence of the pLSM1 cDNA
The pLSM1 cDNA was sequenced and shown to contain an open reading frame of 331 amino acids (Fig. 2). The derived amino acid sequence encodes a protein ap-
LIVINGSTON
FIG.
1. Iv sit71 hybridizations.
Sectioned
ET AL.
gastrula-stage
LUtechinus
embryo
proximately 74% homologous with the corrected deSM50 protein (Karived sequence of the S purpuratus toh-Fukui et al., 1991) and with the derived sequence of a homologue.3 The derived protein seH. pulcherrimus quence also predicts that the protein encoded by pLSM1 is secreted, not glycosylated, has a basic p1 (8.5), and would have a processed molecular weight of 34 kDa after removal of the signal peptide. We propose to call the protein LSM34. The protein also contains in its carboxy portion a tandemly arranged direct repeat of six or seven amino acids that makes up 30% of the protein. A similar fundamental repeat unit is observed in the other two species whose cDNAs have been examined, but there have been some conservative changes in the terminal amino acid of the repeat, and the number of repeats is increased in S. pupuratus and H. pulcherrimus (Fig. 3). Secretion
in Xenopus
Oocytes
The size of the protein encoded by pLSM1 and its ability to be secreted were tested using a Xenopus oocyte translation system. Capped sense transcripts were prepared in vitro from the pLSM1 cDNA and injected into Xenopus oocytes, along with [35S]methionine. The oocytes were incubated at 22°C for 48 hr. The culture medium surrounding injected and control uninjected oo’ T. Higashinakagawa,
personal
communication.
pi&us
probed
S’piclrle
with
Matrix
3H-labeled
ProtfGu
pLSM
probe.
(A)
Light
field;
(B) dark
field.
cytes was removed, and secreted proteins recognized by antibodies against total spicule matrix protein of S. purpuratus were isolated by immuno precipitation. The results of the immunoprecipitations were analyzed by SDS-PAGE and visualized by fluorography (Fig. 4). The results show clearly that the expected 34-kDa protein is secreted by the injected oocytes. Immunoblot
Analysis of Lytechinus Spicule
Matrix
Proteins Antibodies raised in rabbits against total spicule matrix from S. purpuratus and against an SM50 fusion protein (Richardson et al., 1989) were used to analyze the proteins that comprise the organic matrix of the skeletal spicule from Lytechinus pi&us. Spicules were isolated from 4-day-old plutei and demineralized as described under Materials and Methods. The matrix proteins were then separated by SDS-PAGE, transferred to nitrocellulose, and probed separately with the two antibodies. The results are shown in Fig. 5. The antibody to total spicule matrix shows that the L. pictus spicule matrix is composed of a number of cross-reacting proteins (Fig. 5, lanes 2 and 3) that are similar in size range to those that make up the S. purpuratus spicule matrix (Fig. 5, lane 1). Missing, however, is a band corresponding to the approximately 44-kDa SM50 protein seen in extracts prepared from S. purpuratus spicules (Fig. 5, lanes 1 and 4). Using antibodies against an SM50 fusion
476
DEVELOPMENTALBIOLOGY
VOLUME
148,199l
A LSM34 GCACGAGGAA
ATATTGTACC
TTG CTT ATT L L -I TAC Y
TAT Y
GTC CTT L L!
CAGATTGCCA
TTG GCT AGT L A S
CAGGTTTGAG
CTT GTA GCC ATT L V A I
GCT ACA A T
CTT L
GGT CAA G Q
GAC D
TGC CCT C P
AGA R
TAC Y
TTC F
AAC N
CAC H
GCA A
TCT S
GAG TTT TGT GAA E F C E
GTT ACT V T
CCT P
TGT GGA AAC C G N
CGT ATG R M
GTC TAC V -Y
CGA R
ATG M
GTG GCA AGC V A S
GCC A
TGG CTN W L
GGA TGG AAC G W N
CCA P
AAT N
ACT CAA T Q
CCT ATC
GAG AAC
CAT
TTC F
TCG CAA S Q
GAC AAC D N
CAG ATG Q M
GAA E
AAC N
GAA E
AGC S
CCT P
TTC TGG GAA F W E
GAT D
GGT ACC G T
CCT P
TAT Y
ACC T
TCC S
TGG W
GTT AAC V N
CCT P
GCA AAC A N
GGT CAG G Q -
CAC H
GTA ACC V T
GAG TGG GAC E D -W
CTA L
GTG GAG V E
AAC N
ATC I
AGA R
CCG P
GGT ATG G M
CCA 22
TGG ACA W T
CCA P
CCT P
CCA P
CCG P
CAA Q
CAA Q
CCA P
GGT CAA G Q
GCA A
ACT T
GCC A
ATG M
CGT GCT TTT GTC TGT GAG GTA R A F V C E V
CCT P
GGC TTC G F
GGT GGG CAA G G Q
CGA R
CAA Q
GGC TTC G F
CCT P
TTT GGG GGA CAA F G G Q
CAA Q
ACA T
CAA Q
CCT P
AAT N
CCT P
CCT P
GGC TTT GGG GGG CAA G F G G Q
CAA Q
CAG Q
MC N
AGT S
AGG TTC AAC R F N
GAT D
GTT ACA V T
AAC N
CCG P
GGA TCA T G S
CCT P
GGG AGT G S
AAG K
GCT GCA A A
GGG CAA G Q
CAA Q
GGC CAA G Q
CCT P
GGC TTC G F
GGC TTC GGC GGG CAA G F G G Q
GGC TTC GGC GGG CAA G F G G Q
CAG CCC Q P
CCA P
GTC ATG V M
CTT GGA CCA L G P
GGC TTC G F
GGG GGG CGA CAA G G R Q
CAG TCA Q S -
AGT GGA TGG CCC S G W P
GGT AGG GCT CCC G R A P CCA P
CGC R
TCG TCC
TTC GCT GGC TTT CAC F A G F H -
CGA R
-
ATA
GGA G + TCG TGG AGA CCT S W R P
CCT P
ATG M
ATT I
GCA A
GGT GCG TCA TGT TAC G A S C Y
GAA -E
GAA ACT E T
GGA G
TCC S
GGT GCT CTA GCT TCA
CCA P
AAG K
AGC S
ATG
r
+ ATG M
GTC CGG AGT V R S
GGT CCC TCT AGA USRMGALASISSPIENH
GCT TAT A Y
ACGGAACG
CAA Q
CCT GGC TTC P G F
GGC TTT GGC GGG CAG G F G G Q
AGA R
AAGACTCGAA
CCA P
CGT ATG R M
CAA Q
CCC P
GGG GGG G G CCT P
GGC G
GGC GGG CAA G G Q
CAG Q
CCT P
GGC TTT G F
CTC L
CAG Q
GAA GCT E A
CTCAGATTGA
ACTTACTACT
CTCACATTGT
TATTTTTAAT
TTCTCTTCAA
CTATGTGTTT
ATATTCCTAC
ATCATAAACC
GGATTTTGAT
GACTCTGTGA
ACACCAAATT
GTTTTAGGGT
GCCCTCAACG
CAATGTTCCA
TGTTCAATCT
GNGAAAGCAC
TTCTTGGTAT
TTTTGCAACA
GTGTTAAAGG
AGCGATGTGG
TTTGTGCAGG
TACGCTATCG
GAGAACAGTT
ACAGAATACA
AGCCTAGAAG
GATAACATAC
GTTTGAACAA
TCTGTTTGAA
TTATTGATAC
TAAAGGGTCT
ATGCCTTAGC
CGGTCTATTC
ATTTTGTGGC
ATAGGCCTAC
TTAAATTTGT
TTTCTTTTTT
GAAAAGGACA
TTGGATATGA
TTGTAAAGAT
TTAAATTTTG
AATCACGATG
AAGTTAGTAA
AATAAATCTC
GTGCC
B 1
1499
FIG. 2. (A) Sequence of the pLSM cDNA. Amino acids homologous with 5’. ;uurpurutus SM50 are underlined. Arrows show the start of the signal sequence and proline-rich region. The repeat region is boxed. (B) Sequencing strategy. In the coding region, 80% of both strands were sequenced. Overall, 55% of both strands were sequenced. Arrows denote the direction and length of the strands sequenced.
Lytechims
LIVINGSTONETAL.
pictus Spicule Muiris Protein
477
A. LSM34 Repeat
Proline
Signal
SM50 Proline
Signal
Repeat
B. LSM34
G
G
Q(R)
Q
P
G
F
SM50
G
G
R(Q)
Q
P
G
FOf,W
(G)
G
--
V
P
G
V
G
Q
Q
P
G
ELASTIN GLUTENIN
FIG. 3. (A) Comparison of the protein organization of S. ~r~~~~rtrtus and L!/techitrus spicule matrix proteins. (B) Comparison of the similar repeat domains of LSM34, SM50, chick elastin, and wheat glutenin. Comparisons were made using the Intelligenetics IFind program, utilizing the Wilbur and Lipman (1983) algorithm and the default parameters; window = 20, word length protein = 2, density protein = less, fast protein = yes, gap size penalty = 2
protein, it was determined that the L. pictus protein homologue to SM50 has an approximately molecular weight of 34 kDa (Fig. 5, lane 5). This is in agreement with the size of protein predicted by the nucleotide sequence of the cDNA, as well as with the size of the protein secreted by Xenopus oocytes injected with pLSM transcripts. Glycos ylation of LSM34
In order to determine if LSM34 is glycosylated, total spicule matrix proteins were isolated from L. pictus. Half of the supernatant was treated with glycopeptidase F for 18 hr at 37°C. The control half was incubated at the same temperature without the addition of enzyme. The proteins were separated by SDS-PAGE, and LSM34 was detected by immunoblotting using the antiSM50 antibody. The results are shown in Fig. 6. There is no difference in size between the treated (lane 2) and untreated (lane 1) proteins, indicating that the protein is not glycosylated. Control lanes of treated glycoproteins confirmed that the enzyme was active (data not shown). Similar experiments using proteins synthesized in Xenopus oocytes and using S. purpuratus spicule matrix proteins gave the same result (data not shown). DISCUSSION
The skeletal spicule of echinoderms develops within a syncytial cytoplasmic cable formed by pseudopodial projections of primary mesenchyme cells (Gibbins et al.,
1969; Okazaki, 1975). The proteins that make up the matrix of the spicule are believed to be secreted into a domain of extracellular space almost completely surrounded by this syncytium (Decker et al., 1987); the proteins then assemble into a series of concentric lamellae connected to one another by struts (Benson et al., 1983). The matrix is then thought to aid in CaCO, deposition by nucleation and growth modulation (Weiner, 1986). The organic matrices of the mineralized tissues examined to date have been found to be composed of two types of molecules; soluble, acidic, hydrophilic (glyco) proteins associated with the mineral phase, and hydrophobic proteoglycan framework constituents (Weiner, 1986). Bulk analysis of the amino acid and carbohydrate composition of isolated matrices are consistent with this (Benson et al., 1986). Recently a gene (SM50) encoding a soluble protein component of the spicule matrix from a sea urchin, S purpuratus, was isolated, and the predicted amino acid sequence of the protein encoded by this gene was reported to fit the expected profile (Sucov et al., 1987). The present work documents a cDNA from L. picks that encodes a homologue of SM50. The temporal and spatial expression of the RNA during embryogenesis that corresponds to this gene is consistent with its being a component of the spicule matrix. The predicted amino acid sequence of the L. pictus protein (LSM34), as well as that of a homologue from Hemicentrotus pulcherrimus, suggested that the proteins are actually basic and not glycosylated. We have taken advantage of cross-react-
478
DEVELOPMENTALBIOLOGY
I a -
VOLUME 148,1991 Glycopeptidase
I a +
F +
34kd-
34 Kd-
FIG. 6. Glycopeptidase F treatment of LSM34. Spicule matrix proteins were treated with control buffer (p) or glycopeptidase F (+) and analyzed by SDS-PAGE, Western blotting using anti-SM50 antibody, and chemiluminescent detection.
FIG. 4. Secretion from oocytes. Oocytes uninjected (-RNA) or injected (+RNA) were cultured 48 hr. Immunoprecipitates of spicule matrix proteins from 250 pl of culture medium from 25 oocytes were analyzed on a 12.5% SDS-PAGE, followed by fluorography to detect ?S-labeled proteins.
ing antibodies raised against SM50 to examine the characteristics of the LSM34 protein itself. The pLSM cDNA encodes a protein of 34 kDa, after removal of the signal peptide, and this is consistent with
Anti-SM 1
2
3
Anti-WI 4
50 5
i
30.
21-
FIG. 5. Western blot of spicule matrix proteins probed with antibodies to total spicule matrix proteins (l-3) and to SM50 (4,5). Lanes 1,4 = 1 pg S. prrpuratus spicule matrix proteins; lane 2 = 2 ~8, lanes 3,5 = 4 pg L. pict~ks spicule matrix proteins.
what is seen in Western blots of L. pi&s spicule matrix proteins probed with anti-SM50 antibody, and with proteins translated from RNA synthesized in vitro in Xenopus oocytes. The signal peptide encoded by the pLSM cDNA is sufficient for the protein to be secreted by Xenopus oocytes, which is consistent with previous reports (Wilt and Benson, 1988) that synthesis of spicule matrix proteins follows “classical” pathways of secretory proteins from endoplasmic reticulum to Golgi to secretory vesicles. The LSM34 protein does not seem to be glycosylated, since treatment with glycopeptidase F causes no change in mobility. Previous studies have shown that the spicule matrix contains no O-linked carbohydrate residues or N-linked residues of the high mannose type gel analysis of (Benson et al, 1986). Two-dimensional both the LSM34 and SM50 proteins show them to be basic? Sequence comparison between the L. pi&us and S. purpuratus proteins show an overall homology of approximately 74%. While the overall organization of the protein into a signal sequence, proline-rich region, and repeat region is conserved, their is some divergence between the two species. In the proline-rich region, 10 prolines are conserved between the two species. However, there are additional prolines in each cDNA that do not appear in the cDNA of the other species, and the spacing between prolines is not always conserved. In the repeat region, the number of repeats is greater in S. purpuratus (29) than in Lytechinus (14). The amino acid sequence of the first three repeats of the proteins from the two spe4 Chris
Killian,
personal
communication
LIVINGSTON ET AL.
Lgtechinus
ties show 85% homology. Following that, while the type of repeat structure is similar, there is no direct correspondence between the sequences until after the repeat domain. The majority of the repeats in Lytechinus have a glutamine in the third position of the repeat, while the majority of repeats in S. purpuratus have an arginine in the corresponding position. The terminal nonpolar amino acid in the Lytechinus repeat is always a phenylalanine, while in S. purpuratus it can be any one of three nonpolar amino acids. Two independently isolated cDNA clones encoding LSM34 were sequenced, and there were no differences in the amino acid sequence. Taken together, this suggests that the two species shared a common ancestor with a gene containing only a few repeats, and that after the species diverged, the repeats were duplicated to varying degrees. It also suggests that the function of both the proline-rich domain and the repeat domain of these proteins depend more on their three-dimensional structure than on a specific stretch of amino acid sequence. Comparison to protein databases reveals that the repeat regions of these proteins have some similarity to the repeat regions of two classes of proteins, elastins, and wheat glutenins. The repeats in all of these proteins are direct, reiterated many times, and are not regular in the number of amino acids between repeats. The unifying feature of all three of these repeats is that they are predicted to form ,&turn structures (Chou and Fasman, 1978). The repeat regions of both elastin (Urry, D. W., 1982) and glutenin (Miles et ah, 1991) have been shown to form an unusual P-spiral supersecondary structure. This region has been implicated in conferring elastic properties to both molecules. The structure of the LSM34 protein suggests it takes on an elongated shape, with an elastic, P-spiral domain at its carboxy end. The basic nature of the LSM34 protein may allow it to interact with the other, acidic, components of the sea urchin spicule matrix. Future studies on the structure and function of this protein could provide interesting information on the process of biomineralization. We thank Susan Roberts for injecting oocytes, Steve Benson for providing anti-SM50 antibody, and Chris Killian for critically reading the manuscript. This work was supported by NIH Research Grant HD15043 and NASA Research Grant NAG 2-572 to F.W. and by California Cancer Research Coordinating Committee postdoctoral fellowship to B.T.L. REFERENCES BENSON, S. C., JONES, E. M. E., CRISE-BENSON, N., and WILT, F. (1983). Morphology of the organic matrix of the spicule of sea urchin larvae. Exp. Cell Res. 148, 249-253. BENSON, S. C., CRISE-BENSON, N., and WILT, F. (1986). The organic matrix of the skeletal spicule of sea urchin embryos. J. Cell Biol. 102, 1878-1886. BENSON, S. C., Sucov, H. M., STEPHENS, L., DAVIDSON, E. H., and
picks
Spicule
Mutriz
479
Protein
WILT, F. (1987). il lineage-specific gene encoding a major matrix protein of the sea urchin embryo spicule. Dev. Biol. 120,499-506. CHOU, P. Y., and FASMAN, G. D. M. (1978). Prediction of the secondary structure of proteins from their amino acid sequence. Adn Enzymol. 47, 45.
Cox, K. H., ANCERER, L. M., LEE, J. J., DAVIDSON, E. H., and ANGERER, R. C. (1984). Cell lineage-specific programs of expression of multiple actin genes during sea urchin embryogenesis. J Mol. Biol. 188,159172. DECKER, G. L., MORRILL, J. B., and LENNARZ, W. J. (1987). Characterization of sea urchin primary mesenchyme cells and spicules during biomineralization in vitro. Dewlopment 101, 297-312. DREYFUS, G., ADAM, S. A., and CHOI, Y. D. (1984). Physical change in cytoplasmic messenger ribonucleoprotein in cells treated with inhibitors of mRNA transcription. Mol. Cell Biol. 4, 415-423. GIBBINS, J. R., TILNEY, L. G., and PORTER, K. R. (1969). Microtubules in the formation and development of the primary mesenchymein Arabacia
pw~ctulata.
.J. Cell Biol.
41, 201-226.
HALL, H. G. (1978). Hardening of the sea urchin fertilization envelope by peroxidase-catalyzed phenolic coupling of tyrosine. Cell 15,343355. HARKEY, M. A., and WHITELY, A. H. (1983). The program of protein synthesis during the development of the micromere-primary mesenthyme cell line in the sea urchin embryo. Dev. Biol. 100, 12-28. HINEGARDNER, R. (1967). Echinoderms. In “Methods in Developmental Biolo&’ (F. Wilt and N. K. Wessels, Eds.), pp. 139-155. T. Y. Crowell, New York. JOHNSON, D., GAUTSCH, J. W., SPORTSMAN, J. R., and ELDER, J. H. (1984). Improved technique utilizing nonfat dry milk for analysis of protein and nucleic acid transferred to nitrocellulose. Ge~lef. Anal. Tech nol. 1, 3-8.
KATOH-FUKUI, Y., NOCE, T., UEDA, T., FUJIWARA, Y., HASHIMOTO, N., HIGASHINAKAGAWA, T., KILLIAN, C. E., LIVINGSTON, B. T., WILT, F. H., BENSON, S. C., Sucov. H. M., and DAVIDSON, E. H. (1991). The corrected structure of the SM50 spicule matrix protein of Strcny~yltr centrotrrs purpurc~tus Del: Biol., in press. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature fLowdow) 227, 680685. LEAF, D. S., ROBERTS, S. J., GERHARDT, J. C., and HSIAO-PING MOORE. (1990). The secretory pathway is blocked between the trans-golgi and the plasma membrane during meiotic maturation in X~?ro~nr,s oocytes. De73. Biol. 141, l-12. MARCH, C. J., MOSLEY, B., CARSEN, A., CERRETI, D. P., BRAEDT, G., PRICE, V., GILLIS, S., HENNEY, C. S., KRONHEIM, S. R., GRABSTEIN, K., CONLON, P. J., HOPP, T. P., and COSMAN, D. (1985). Cloning, sequencing and expression of two distinct human interleukin-1 complementary DNAs. Nature (Londo1~) 315, 641-647. MELTON, D., KRIEC, P., REBAGLIATI, M., MANIATUS, T., ZINN, K., and GREEN, M. (1984). Efficient i71 vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promotor. Nucleic Acids Res. 12,7035-7056. MILES, M. J., CARR, H. J., MCMASTER, T. C., I’ANSON, K. J., BELTON, P. S., MORRIS, V. J., FIELD, J. M., SHEWRY, P. R., and TATHAM, A. S. (1991). Scanning tunneling microscopy of a wheat seed storage protein reveals details of an unusual supersecondary structure. t-‘roc Natl.
Acad.
Sci. USA 88, 68-71.
MOORE, H. P., and KELLY, R. (1985). Secretory protein targeting in a pituitary cell line: Differential transport of foreign secretory proteins to distinct secretory pathways. J. Cell Biol. 101, 1773-1781. OKAZAKI, K. (1975) Spicule formation by isolated micromeres of the sea urchin embryos. ,4mer. Zool. 15, 567-581. RICHARDSON, W., KITAJIMA, T., WILT, F., and BENSON, S. C. (1989). Expression of an embryonic spicule matrix gene in calcified tissues of adult sea urchins. De/%. Biol. 132, 266-269.
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DEVELOPMENTAL BIOLOGY
SAMBROOK, J., FRITSCH, E. F., and MANIATIS, T. (1989). In “Molecular Cloning: A Laboratory Manual.” Cold Springs Harbor Laboratory Press, Cold Springs Harbor, NY. STEPHENS, L., HARDIN, J., KELLER, R., and WILT, F. (1989). The effects of aphidicolin on morphogenesis and differentiation in the sea urchin embryo. Dev. Biol. 118,64-69. Sucov, H. M., BENSON, S. C., ROBINSON,J. J., BRITTEN, R. J., WILT, F., and DAVIDSON, E. H. (1987). A lineage-specific gene encoding a major matrix protein of the sea urchin embryo spicule. II. A structure of the gene and derived sequence of the protein. Dev. Biol. 120,507 519. TOWBIN, H., STACHELIN, T., and GORDON, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76,4350-4354.
VOLUME 148,1991
URRY, D. W. (1982). Characterization of soluble peptides of elastin by physical techniques. In “Methods in Enzymology: Structural and Contractile Proteins, Part A, Extracellular Matrix” (L. W. Cunningham and D. W. Frederiksen, Eds.), Vol. 82, pp. 673-716. Academic Press, New York. WEINER, S. (1984). Organization of organic matrix materials in mineralized tissues. Am. 2001. 24, 945-952. WEINER, S. (1986). Organization of extracellularly mineralized tissues: a comparative study of biological crystal growth. CRC Crit. Rev. Biochem.
20, 365-408.
WILBUR, W. J., and LIPMAN, D. J. (1983). Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA 80, 726-739.
WILT, F., and BENSON, S. (1988). “Self-Assembling Alan R. Liss, Inc., New York.
Architecture.”
148,47%48u
(19%)
Characterization of a cDNA Encoding a Protein Involved in Formation of the Skeleton during Development of the Sea Urchin Lytechinus pictus BRIAN Department
of Molecular
T. LIVINGSTON,’ and
Cell
Biology,
ROBIN SHAW, 5’7.9 La&
Sciences
ADINA
Addition,
BAILEY, Uniwrsity
AND FRED WILT of Culifbrnia,
Berkeley,
Cula&Gz
9~720
In order to investigate the role of proteins in the formation of mineralized tissues during development, we have isolated a cDNA that encodes a protein that is a component of the organic matrix of the skeletal spicule of the sea urchin, Lytechinus fictus. The expression of the RNA encoding this protein is regulated over development and is localized to the descendents of the micromere lineage. Comparison of the sequence of this cDNA to homologous cDNAs from other species of urchin reveal that the protein is basic and contains three conserved structural motifs: a signal peptide. a proline-rich region, and an unusual region composed of a series of direct repeats. Studies on the protein encoded by this cDNA confirm the predicted reading frame deduced from the nucleotide sequence and show that the protein is secreted and not glycosylated. Comparison of the amino acid sequence to databases reveal that the repeat domain is similar to cc>19Y1 Academic Press, Inc. proteins that form a unique B-spiral supersecondary structure. INTRODIJCTION
The role of proteins that participate in the formation of mineralized tissue is poorly understood, largely due to the complex nature of the tissues studied. Isolation and characterization of the protein components of mineralized structures are important first steps in elucidating their function. The endoskeletal spicule of sea urchin larvae has provided a simple system to begin the study of individual proteins involved in the formation of mineralized tissue during development. The spicule is formed during embryonic development by a group of cells, called micromeres, which arise at the vegetal pole of the embryo during the fourth cleavage division. These cells give rise to the primary mesenchyme cells, which migrate into the blastocoel cavity and in turn differentiate into the skeletal spicules (Wilt and Benson, 1988). The organic matrix of the spicule has been isolated (Benson et al., 1986), and studies of the matrix have shown that it is composed of a small number of proteins that are predicted to be highly acidic, N-linked glycoproteins. This is in agreement with the nature of organic matrices found in other mineralized tissues (Weiner, 1984). cDNA and genomic clones that encode a spicule matrix protein (SM50) were first isolated from the sea urchin Stmngylocentrotus purpuratus. The amino acid sequence of the protein predicted by the nucleotide sequence of the gene was initially reported to be acidic and to contain sequences involved in secretion and N-linked
’ To whom
correspondence
should
glycosylation (Sucov et o,l, 1987). However, sequence information from homologous cDNAs from two other species, Hemicentrotus’ and Lytechinus picks have revealed an error in the nucleotide sequence of the SM50 gene (Katoh-Fukui et ub, 1991). The new derived amino acid sequence for the SM50 protein and its homologues now predicts them to be basic, nonglycosylated proteins. We report here the cloning and characterization of a cDNA (pLSM1) from Lytech,inus p&us encoding a protein, LSM34, that is the homologue of SM50, the S. purpuratus gene encoding a spicule matrix protein. The characterization of a homologous cDNA from a species that diverged from S purpuratus some 60 million years ago has allowed us to examine the sequence for conserved regions that may encode important functional domains, and to infer how the two genes have evolved from a common ancestor. We have also begun to characterize the protein (LSM34) itself. The 34-kDa protein is secreted, is not glycosylated, and contains several conserved domains, one of which contains an unusual six- to seven-amino acid repeat. MATERIALS
473
METHODS
Culturing Sea Urchin Embryos L. pictus gametes were obtained, eggs were fertilized, and embryos were cultured according to conventional methods (Hinegardner, 1967). Fertilization membranes were removed as described by Hall (1978).
’ T. Higashinakagawa,
be addressed.
AND
personal
communication. OOIZ-1606/91 Copyright All rights
$3.00
c 1991 by Academic Press, Inc. of reproduction in any form reserved.
474
DEVELOPMENTALBIOLOGY V0~~~~148,1991
Isolation
of RNA
Mesenchyme and endoderm cell fractions were isolated from pluteus-stage larvae using a procedure described by Harkey and Whitely (1980). RNA was isolated from these cells using the method of March et al. (1985) as modified by Brandhorst (personal communication) and described by Stephens et al. (1989). Poly(A)+ RNA was isolated on oligo-dT cellulose columns as described by Sambrook et al. (1989). RNA Probe Synthesis
Single stranded RNA probes for SM50, labeled with [32P]UTP, (800 Ci/mmole) were synthesized from the pHS72 plasmid (Benson et al., 1987) using the SP6 promoter of pSP65 (Melton et al., 1984). Probes for pLSM were synthesized using rH]UTP from the T3 promoter of Bluescript (Stratagene), using the cloned cDNA described herein. XgtlO
Library
Construction
and Screening
cDNA was synthesized from 5 pug of poly(A)+ RNA using Pharmacia’s cDNA synthesis system according to the manufacturers instructions. EcoRI linkers were added, the cDNA was ligated into the EcoRI site of XgtlO, and packaged into phage particles using Gigapack Gold (Stratagene) packaging extracts, as described by the manufacturer. Phage were plated and transferred to nitrocellulose using standard procedures (Sambrook et al., 1989). Hybridizations using the SM50 RNA probe were at 60°C in 4~ SET buffer (80 mM Tris (pH 8.0), 0.6 M NaCl, 4 mM EDTA), 5~ Denhardts, and 100 yg yeast RNA/ml for 18 hr. Filters were washed three times at 60°C in 0.3~ SET and positive plaques detected by autoradiography. In Situ Hybridizations In situ hybridizations were carried out as described by Cox et al. (1984) using [3H]UTP-labeled probes. Emulsion coated slides were exposed for 14 days. DNA Sequencing
DNA was sequenced using Sequenase (USB) sequencing system according to standard dideoxy-sequencing methods. Translation in Xenopus Oocytes
Full length, capped, sense strand RNA was synthesized from the pLSM1 cDNA, subcloned into the EcoRI site of pGEM4Z (Promega), using SP6 polymerase, as described by Sambrook et al. (1989). Oocytes were obtained, injected, and cultured as described by Leaf et al.
(1990). [35S]Methionine (40-50 nl, 0.6-0.75 PCi) was injected into oocytes to label newly synthesized proteins. Culture medium was removed after 48 hr, and immunoprecipitations were performed according to Moore and Kelly (1985), using antibody raised against the total spicule matrix of S. purpuratus (Benson et al, 1986). Electrophoresis was in 12.5% acrylamide gels as described by Laemmli (1970) and modified by Dreyfus et al. (1984). Immunoblotting
Electrophoresis was done as described above. Immunoblotting was done as described by Towbin et al. (1979), with modifications described by Johnson et al. (1984). Binding of antibody to proteins on the nitrocellulose was carried out using a chemiluminescent detection system (Amersham) as described by the manufacturer. Gtycosidase Digestions
Glycopeptidase F (Boehringer-Mannheim) digestions were carried out at 37°C for 18 hr, as described by the manufacturer. Electrophoresis was carried out as described above. RESULTS Developmental Expression
The cDNA clone pLSM1 was isolated from a L. pictus XgtlO cDNA library using an S. purpwatus cDNA, pHS72, known to encode a spicule matrix protein, as probe. The timing of the expression of this cDNA was examined using Northern blot analysis. Total RNA from various stages of development was subjected to electrophoresis, blotted to nitrocellulose, and probed with radioactive transcripts from the cDNA. The RNA was first expressed at blastula and continued to be detected through the pluteus stage (data not shown). This is consistent with the pattern of expression seen in S. purpuratus (Benson et al., 1987). The cellular localization of transcripts was examined using in situ hybridization with 3H RNA probes transcribed from the cDNA clone of L. pi&s. Figure 1 shows a representative sample of the results. The transcripts are localized exclusively in the primary mesenchyme cells within the blastocoel. Again this is consistent with the expression of the S. pwpuratus homologue, and suggests the pLSM1 cDNA encodes a spicule matrix protein. Sequence of the pLSM1 cDNA
The pLSM1 cDNA was sequenced and shown to contain an open reading frame of 331 amino acids (Fig. 2). The derived amino acid sequence encodes a protein ap-
LIVINGSTON
FIG.
1. Iv sit71 hybridizations.
Sectioned
ET AL.
gastrula-stage
LUtechinus
embryo
proximately 74% homologous with the corrected deSM50 protein (Karived sequence of the S purpuratus toh-Fukui et al., 1991) and with the derived sequence of a homologue.3 The derived protein seH. pulcherrimus quence also predicts that the protein encoded by pLSM1 is secreted, not glycosylated, has a basic p1 (8.5), and would have a processed molecular weight of 34 kDa after removal of the signal peptide. We propose to call the protein LSM34. The protein also contains in its carboxy portion a tandemly arranged direct repeat of six or seven amino acids that makes up 30% of the protein. A similar fundamental repeat unit is observed in the other two species whose cDNAs have been examined, but there have been some conservative changes in the terminal amino acid of the repeat, and the number of repeats is increased in S. pupuratus and H. pulcherrimus (Fig. 3). Secretion
in Xenopus
Oocytes
The size of the protein encoded by pLSM1 and its ability to be secreted were tested using a Xenopus oocyte translation system. Capped sense transcripts were prepared in vitro from the pLSM1 cDNA and injected into Xenopus oocytes, along with [35S]methionine. The oocytes were incubated at 22°C for 48 hr. The culture medium surrounding injected and control uninjected oo’ T. Higashinakagawa,
personal
communication.
pi&us
probed
S’piclrle
with
Matrix
3H-labeled
ProtfGu
pLSM
probe.
(A)
Light
field;
(B) dark
field.
cytes was removed, and secreted proteins recognized by antibodies against total spicule matrix protein of S. purpuratus were isolated by immuno precipitation. The results of the immunoprecipitations were analyzed by SDS-PAGE and visualized by fluorography (Fig. 4). The results show clearly that the expected 34-kDa protein is secreted by the injected oocytes. Immunoblot
Analysis of Lytechinus Spicule
Matrix
Proteins Antibodies raised in rabbits against total spicule matrix from S. purpuratus and against an SM50 fusion protein (Richardson et al., 1989) were used to analyze the proteins that comprise the organic matrix of the skeletal spicule from Lytechinus pi&us. Spicules were isolated from 4-day-old plutei and demineralized as described under Materials and Methods. The matrix proteins were then separated by SDS-PAGE, transferred to nitrocellulose, and probed separately with the two antibodies. The results are shown in Fig. 5. The antibody to total spicule matrix shows that the L. pictus spicule matrix is composed of a number of cross-reacting proteins (Fig. 5, lanes 2 and 3) that are similar in size range to those that make up the S. purpuratus spicule matrix (Fig. 5, lane 1). Missing, however, is a band corresponding to the approximately 44-kDa SM50 protein seen in extracts prepared from S. purpuratus spicules (Fig. 5, lanes 1 and 4). Using antibodies against an SM50 fusion
476
DEVELOPMENTALBIOLOGY
VOLUME
148,199l
A LSM34 GCACGAGGAA
ATATTGTACC
TTG CTT ATT L L -I TAC Y
TAT Y
GTC CTT L L!
CAGATTGCCA
TTG GCT AGT L A S
CAGGTTTGAG
CTT GTA GCC ATT L V A I
GCT ACA A T
CTT L
GGT CAA G Q
GAC D
TGC CCT C P
AGA R
TAC Y
TTC F
AAC N
CAC H
GCA A
TCT S
GAG TTT TGT GAA E F C E
GTT ACT V T
CCT P
TGT GGA AAC C G N
CGT ATG R M
GTC TAC V -Y
CGA R
ATG M
GTG GCA AGC V A S
GCC A
TGG CTN W L
GGA TGG AAC G W N
CCA P
AAT N
ACT CAA T Q
CCT ATC
GAG AAC
CAT
TTC F
TCG CAA S Q
GAC AAC D N
CAG ATG Q M
GAA E
AAC N
GAA E
AGC S
CCT P
TTC TGG GAA F W E
GAT D
GGT ACC G T
CCT P
TAT Y
ACC T
TCC S
TGG W
GTT AAC V N
CCT P
GCA AAC A N
GGT CAG G Q -
CAC H
GTA ACC V T
GAG TGG GAC E D -W
CTA L
GTG GAG V E
AAC N
ATC I
AGA R
CCG P
GGT ATG G M
CCA 22
TGG ACA W T
CCA P
CCT P
CCA P
CCG P
CAA Q
CAA Q
CCA P
GGT CAA G Q
GCA A
ACT T
GCC A
ATG M
CGT GCT TTT GTC TGT GAG GTA R A F V C E V
CCT P
GGC TTC G F
GGT GGG CAA G G Q
CGA R
CAA Q
GGC TTC G F
CCT P
TTT GGG GGA CAA F G G Q
CAA Q
ACA T
CAA Q
CCT P
AAT N
CCT P
CCT P
GGC TTT GGG GGG CAA G F G G Q
CAA Q
CAG Q
MC N
AGT S
AGG TTC AAC R F N
GAT D
GTT ACA V T
AAC N
CCG P
GGA TCA T G S
CCT P
GGG AGT G S
AAG K
GCT GCA A A
GGG CAA G Q
CAA Q
GGC CAA G Q
CCT P
GGC TTC G F
GGC TTC GGC GGG CAA G F G G Q
GGC TTC GGC GGG CAA G F G G Q
CAG CCC Q P
CCA P
GTC ATG V M
CTT GGA CCA L G P
GGC TTC G F
GGG GGG CGA CAA G G R Q
CAG TCA Q S -
AGT GGA TGG CCC S G W P
GGT AGG GCT CCC G R A P CCA P
CGC R
TCG TCC
TTC GCT GGC TTT CAC F A G F H -
CGA R
-
ATA
GGA G + TCG TGG AGA CCT S W R P
CCT P
ATG M
ATT I
GCA A
GGT GCG TCA TGT TAC G A S C Y
GAA -E
GAA ACT E T
GGA G
TCC S
GGT GCT CTA GCT TCA
CCA P
AAG K
AGC S
ATG
r
+ ATG M
GTC CGG AGT V R S
GGT CCC TCT AGA USRMGALASISSPIENH
GCT TAT A Y
ACGGAACG
CAA Q
CCT GGC TTC P G F
GGC TTT GGC GGG CAG G F G G Q
AGA R
AAGACTCGAA
CCA P
CGT ATG R M
CAA Q
CCC P
GGG GGG G G CCT P
GGC G
GGC GGG CAA G G Q
CAG Q
CCT P
GGC TTT G F
CTC L
CAG Q
GAA GCT E A
CTCAGATTGA
ACTTACTACT
CTCACATTGT
TATTTTTAAT
TTCTCTTCAA
CTATGTGTTT
ATATTCCTAC
ATCATAAACC
GGATTTTGAT
GACTCTGTGA
ACACCAAATT
GTTTTAGGGT
GCCCTCAACG
CAATGTTCCA
TGTTCAATCT
GNGAAAGCAC
TTCTTGGTAT
TTTTGCAACA
GTGTTAAAGG
AGCGATGTGG
TTTGTGCAGG
TACGCTATCG
GAGAACAGTT
ACAGAATACA
AGCCTAGAAG
GATAACATAC
GTTTGAACAA
TCTGTTTGAA
TTATTGATAC
TAAAGGGTCT
ATGCCTTAGC
CGGTCTATTC
ATTTTGTGGC
ATAGGCCTAC
TTAAATTTGT
TTTCTTTTTT
GAAAAGGACA
TTGGATATGA
TTGTAAAGAT
TTAAATTTTG
AATCACGATG
AAGTTAGTAA
AATAAATCTC
GTGCC
B 1
1499
FIG. 2. (A) Sequence of the pLSM cDNA. Amino acids homologous with 5’. ;uurpurutus SM50 are underlined. Arrows show the start of the signal sequence and proline-rich region. The repeat region is boxed. (B) Sequencing strategy. In the coding region, 80% of both strands were sequenced. Overall, 55% of both strands were sequenced. Arrows denote the direction and length of the strands sequenced.
Lytechims
LIVINGSTONETAL.
pictus Spicule Muiris Protein
477
A. LSM34 Repeat
Proline
Signal
SM50 Proline
Signal
Repeat
B. LSM34
G
G
Q(R)
Q
P
G
F
SM50
G
G
R(Q)
Q
P
G
FOf,W
(G)
G
--
V
P
G
V
G
Q
Q
P
G
ELASTIN GLUTENIN
FIG. 3. (A) Comparison of the protein organization of S. ~r~~~~rtrtus and L!/techitrus spicule matrix proteins. (B) Comparison of the similar repeat domains of LSM34, SM50, chick elastin, and wheat glutenin. Comparisons were made using the Intelligenetics IFind program, utilizing the Wilbur and Lipman (1983) algorithm and the default parameters; window = 20, word length protein = 2, density protein = less, fast protein = yes, gap size penalty = 2
protein, it was determined that the L. pictus protein homologue to SM50 has an approximately molecular weight of 34 kDa (Fig. 5, lane 5). This is in agreement with the size of protein predicted by the nucleotide sequence of the cDNA, as well as with the size of the protein secreted by Xenopus oocytes injected with pLSM transcripts. Glycos ylation of LSM34
In order to determine if LSM34 is glycosylated, total spicule matrix proteins were isolated from L. pictus. Half of the supernatant was treated with glycopeptidase F for 18 hr at 37°C. The control half was incubated at the same temperature without the addition of enzyme. The proteins were separated by SDS-PAGE, and LSM34 was detected by immunoblotting using the antiSM50 antibody. The results are shown in Fig. 6. There is no difference in size between the treated (lane 2) and untreated (lane 1) proteins, indicating that the protein is not glycosylated. Control lanes of treated glycoproteins confirmed that the enzyme was active (data not shown). Similar experiments using proteins synthesized in Xenopus oocytes and using S. purpuratus spicule matrix proteins gave the same result (data not shown). DISCUSSION
The skeletal spicule of echinoderms develops within a syncytial cytoplasmic cable formed by pseudopodial projections of primary mesenchyme cells (Gibbins et al.,
1969; Okazaki, 1975). The proteins that make up the matrix of the spicule are believed to be secreted into a domain of extracellular space almost completely surrounded by this syncytium (Decker et al., 1987); the proteins then assemble into a series of concentric lamellae connected to one another by struts (Benson et al., 1983). The matrix is then thought to aid in CaCO, deposition by nucleation and growth modulation (Weiner, 1986). The organic matrices of the mineralized tissues examined to date have been found to be composed of two types of molecules; soluble, acidic, hydrophilic (glyco) proteins associated with the mineral phase, and hydrophobic proteoglycan framework constituents (Weiner, 1986). Bulk analysis of the amino acid and carbohydrate composition of isolated matrices are consistent with this (Benson et al., 1986). Recently a gene (SM50) encoding a soluble protein component of the spicule matrix from a sea urchin, S purpuratus, was isolated, and the predicted amino acid sequence of the protein encoded by this gene was reported to fit the expected profile (Sucov et al., 1987). The present work documents a cDNA from L. picks that encodes a homologue of SM50. The temporal and spatial expression of the RNA during embryogenesis that corresponds to this gene is consistent with its being a component of the spicule matrix. The predicted amino acid sequence of the L. pictus protein (LSM34), as well as that of a homologue from Hemicentrotus pulcherrimus, suggested that the proteins are actually basic and not glycosylated. We have taken advantage of cross-react-
478
DEVELOPMENTALBIOLOGY
I a -
VOLUME 148,1991 Glycopeptidase
I a +
F +
34kd-
34 Kd-
FIG. 6. Glycopeptidase F treatment of LSM34. Spicule matrix proteins were treated with control buffer (p) or glycopeptidase F (+) and analyzed by SDS-PAGE, Western blotting using anti-SM50 antibody, and chemiluminescent detection.
FIG. 4. Secretion from oocytes. Oocytes uninjected (-RNA) or injected (+RNA) were cultured 48 hr. Immunoprecipitates of spicule matrix proteins from 250 pl of culture medium from 25 oocytes were analyzed on a 12.5% SDS-PAGE, followed by fluorography to detect ?S-labeled proteins.
ing antibodies raised against SM50 to examine the characteristics of the LSM34 protein itself. The pLSM cDNA encodes a protein of 34 kDa, after removal of the signal peptide, and this is consistent with
Anti-SM 1
2
3
Anti-WI 4
50 5
i
30.
21-
FIG. 5. Western blot of spicule matrix proteins probed with antibodies to total spicule matrix proteins (l-3) and to SM50 (4,5). Lanes 1,4 = 1 pg S. prrpuratus spicule matrix proteins; lane 2 = 2 ~8, lanes 3,5 = 4 pg L. pict~ks spicule matrix proteins.
what is seen in Western blots of L. pi&s spicule matrix proteins probed with anti-SM50 antibody, and with proteins translated from RNA synthesized in vitro in Xenopus oocytes. The signal peptide encoded by the pLSM cDNA is sufficient for the protein to be secreted by Xenopus oocytes, which is consistent with previous reports (Wilt and Benson, 1988) that synthesis of spicule matrix proteins follows “classical” pathways of secretory proteins from endoplasmic reticulum to Golgi to secretory vesicles. The LSM34 protein does not seem to be glycosylated, since treatment with glycopeptidase F causes no change in mobility. Previous studies have shown that the spicule matrix contains no O-linked carbohydrate residues or N-linked residues of the high mannose type gel analysis of (Benson et al, 1986). Two-dimensional both the LSM34 and SM50 proteins show them to be basic? Sequence comparison between the L. pi&us and S. purpuratus proteins show an overall homology of approximately 74%. While the overall organization of the protein into a signal sequence, proline-rich region, and repeat region is conserved, their is some divergence between the two species. In the proline-rich region, 10 prolines are conserved between the two species. However, there are additional prolines in each cDNA that do not appear in the cDNA of the other species, and the spacing between prolines is not always conserved. In the repeat region, the number of repeats is greater in S. purpuratus (29) than in Lytechinus (14). The amino acid sequence of the first three repeats of the proteins from the two spe4 Chris
Killian,
personal
communication
LIVINGSTON ET AL.
Lgtechinus
ties show 85% homology. Following that, while the type of repeat structure is similar, there is no direct correspondence between the sequences until after the repeat domain. The majority of the repeats in Lytechinus have a glutamine in the third position of the repeat, while the majority of repeats in S. purpuratus have an arginine in the corresponding position. The terminal nonpolar amino acid in the Lytechinus repeat is always a phenylalanine, while in S. purpuratus it can be any one of three nonpolar amino acids. Two independently isolated cDNA clones encoding LSM34 were sequenced, and there were no differences in the amino acid sequence. Taken together, this suggests that the two species shared a common ancestor with a gene containing only a few repeats, and that after the species diverged, the repeats were duplicated to varying degrees. It also suggests that the function of both the proline-rich domain and the repeat domain of these proteins depend more on their three-dimensional structure than on a specific stretch of amino acid sequence. Comparison to protein databases reveals that the repeat regions of these proteins have some similarity to the repeat regions of two classes of proteins, elastins, and wheat glutenins. The repeats in all of these proteins are direct, reiterated many times, and are not regular in the number of amino acids between repeats. The unifying feature of all three of these repeats is that they are predicted to form ,&turn structures (Chou and Fasman, 1978). The repeat regions of both elastin (Urry, D. W., 1982) and glutenin (Miles et ah, 1991) have been shown to form an unusual P-spiral supersecondary structure. This region has been implicated in conferring elastic properties to both molecules. The structure of the LSM34 protein suggests it takes on an elongated shape, with an elastic, P-spiral domain at its carboxy end. The basic nature of the LSM34 protein may allow it to interact with the other, acidic, components of the sea urchin spicule matrix. Future studies on the structure and function of this protein could provide interesting information on the process of biomineralization. We thank Susan Roberts for injecting oocytes, Steve Benson for providing anti-SM50 antibody, and Chris Killian for critically reading the manuscript. This work was supported by NIH Research Grant HD15043 and NASA Research Grant NAG 2-572 to F.W. and by California Cancer Research Coordinating Committee postdoctoral fellowship to B.T.L. REFERENCES BENSON, S. C., JONES, E. M. E., CRISE-BENSON, N., and WILT, F. (1983). Morphology of the organic matrix of the spicule of sea urchin larvae. Exp. Cell Res. 148, 249-253. BENSON, S. C., CRISE-BENSON, N., and WILT, F. (1986). The organic matrix of the skeletal spicule of sea urchin embryos. J. Cell Biol. 102, 1878-1886. BENSON, S. C., Sucov, H. M., STEPHENS, L., DAVIDSON, E. H., and
picks
Spicule
Mutriz
479
Protein
WILT, F. (1987). il lineage-specific gene encoding a major matrix protein of the sea urchin embryo spicule. Dev. Biol. 120,499-506. CHOU, P. Y., and FASMAN, G. D. M. (1978). Prediction of the secondary structure of proteins from their amino acid sequence. Adn Enzymol. 47, 45.
Cox, K. H., ANCERER, L. M., LEE, J. J., DAVIDSON, E. H., and ANGERER, R. C. (1984). Cell lineage-specific programs of expression of multiple actin genes during sea urchin embryogenesis. J Mol. Biol. 188,159172. DECKER, G. L., MORRILL, J. B., and LENNARZ, W. J. (1987). Characterization of sea urchin primary mesenchyme cells and spicules during biomineralization in vitro. Dewlopment 101, 297-312. DREYFUS, G., ADAM, S. A., and CHOI, Y. D. (1984). Physical change in cytoplasmic messenger ribonucleoprotein in cells treated with inhibitors of mRNA transcription. Mol. Cell Biol. 4, 415-423. GIBBINS, J. R., TILNEY, L. G., and PORTER, K. R. (1969). Microtubules in the formation and development of the primary mesenchymein Arabacia
pw~ctulata.
.J. Cell Biol.
41, 201-226.
HALL, H. G. (1978). Hardening of the sea urchin fertilization envelope by peroxidase-catalyzed phenolic coupling of tyrosine. Cell 15,343355. HARKEY, M. A., and WHITELY, A. H. (1983). The program of protein synthesis during the development of the micromere-primary mesenthyme cell line in the sea urchin embryo. Dev. Biol. 100, 12-28. HINEGARDNER, R. (1967). Echinoderms. In “Methods in Developmental Biolo&’ (F. Wilt and N. K. Wessels, Eds.), pp. 139-155. T. Y. Crowell, New York. JOHNSON, D., GAUTSCH, J. W., SPORTSMAN, J. R., and ELDER, J. H. (1984). Improved technique utilizing nonfat dry milk for analysis of protein and nucleic acid transferred to nitrocellulose. Ge~lef. Anal. Tech nol. 1, 3-8.
KATOH-FUKUI, Y., NOCE, T., UEDA, T., FUJIWARA, Y., HASHIMOTO, N., HIGASHINAKAGAWA, T., KILLIAN, C. E., LIVINGSTON, B. T., WILT, F. H., BENSON, S. C., Sucov. H. M., and DAVIDSON, E. H. (1991). The corrected structure of the SM50 spicule matrix protein of Strcny~yltr centrotrrs purpurc~tus Del: Biol., in press. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature fLowdow) 227, 680685. LEAF, D. S., ROBERTS, S. J., GERHARDT, J. C., and HSIAO-PING MOORE. (1990). The secretory pathway is blocked between the trans-golgi and the plasma membrane during meiotic maturation in X~?ro~nr,s oocytes. De73. Biol. 141, l-12. MARCH, C. J., MOSLEY, B., CARSEN, A., CERRETI, D. P., BRAEDT, G., PRICE, V., GILLIS, S., HENNEY, C. S., KRONHEIM, S. R., GRABSTEIN, K., CONLON, P. J., HOPP, T. P., and COSMAN, D. (1985). Cloning, sequencing and expression of two distinct human interleukin-1 complementary DNAs. Nature (Londo1~) 315, 641-647. MELTON, D., KRIEC, P., REBAGLIATI, M., MANIATUS, T., ZINN, K., and GREEN, M. (1984). Efficient i71 vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promotor. Nucleic Acids Res. 12,7035-7056. MILES, M. J., CARR, H. J., MCMASTER, T. C., I’ANSON, K. J., BELTON, P. S., MORRIS, V. J., FIELD, J. M., SHEWRY, P. R., and TATHAM, A. S. (1991). Scanning tunneling microscopy of a wheat seed storage protein reveals details of an unusual supersecondary structure. t-‘roc Natl.
Acad.
Sci. USA 88, 68-71.
MOORE, H. P., and KELLY, R. (1985). Secretory protein targeting in a pituitary cell line: Differential transport of foreign secretory proteins to distinct secretory pathways. J. Cell Biol. 101, 1773-1781. OKAZAKI, K. (1975) Spicule formation by isolated micromeres of the sea urchin embryos. ,4mer. Zool. 15, 567-581. RICHARDSON, W., KITAJIMA, T., WILT, F., and BENSON, S. C. (1989). Expression of an embryonic spicule matrix gene in calcified tissues of adult sea urchins. De/%. Biol. 132, 266-269.
480
DEVELOPMENTAL BIOLOGY
SAMBROOK, J., FRITSCH, E. F., and MANIATIS, T. (1989). In “Molecular Cloning: A Laboratory Manual.” Cold Springs Harbor Laboratory Press, Cold Springs Harbor, NY. STEPHENS, L., HARDIN, J., KELLER, R., and WILT, F. (1989). The effects of aphidicolin on morphogenesis and differentiation in the sea urchin embryo. Dev. Biol. 118,64-69. Sucov, H. M., BENSON, S. C., ROBINSON,J. J., BRITTEN, R. J., WILT, F., and DAVIDSON, E. H. (1987). A lineage-specific gene encoding a major matrix protein of the sea urchin embryo spicule. II. A structure of the gene and derived sequence of the protein. Dev. Biol. 120,507 519. TOWBIN, H., STACHELIN, T., and GORDON, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76,4350-4354.
VOLUME 148,1991
URRY, D. W. (1982). Characterization of soluble peptides of elastin by physical techniques. In “Methods in Enzymology: Structural and Contractile Proteins, Part A, Extracellular Matrix” (L. W. Cunningham and D. W. Frederiksen, Eds.), Vol. 82, pp. 673-716. Academic Press, New York. WEINER, S. (1984). Organization of organic matrix materials in mineralized tissues. Am. 2001. 24, 945-952. WEINER, S. (1986). Organization of extracellularly mineralized tissues: a comparative study of biological crystal growth. CRC Crit. Rev. Biochem.
20, 365-408.
WILBUR, W. J., and LIPMAN, D. J. (1983). Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA 80, 726-739.
WILT, F., and BENSON, S. (1988). “Self-Assembling Alan R. Liss, Inc., New York.
Architecture.”