Biotechnol. Prog. 1900, 6, 171-177
171
ARTICLES Structural and Functional Repetition in a Marine Mussel Adhesive Protein David R. Filpula, Shwu-Maan Lee, Rebecca P. Link, Susan L. Strausberg, and Robert L. Strausberg' Genex Corporation, 16020 Industrial Drive, Gaithersburg, Maryland 20877
The DOPA-rich polyphenolic protein secreted by the marine mussel Mytilus edulis establishes key chemical linkages in a water-resistant adhesive. Molecular cloning of the gene for this remarkable protein reveals its primary structure as one of the most repetitive proteins identified in the animal kingdom. Expression and purification of polyphenolic proteins from recombinant yeast have provided sufficient material to demonstrate adhesivity of these polypeptides in the laboratory. Adhesive tests reveal a water-resistant bonding capacity of the protein that is dependent on in vitro modification of tyrosine residues to DOPA and the subsequent oxidation to quinone.
Introduction
expression of a cDNA clone designated 14-1that encodes a 24 000 molecular weight carboxy terminal region of the adhesive protein (9). This clone encodes an adhesive protein carrying 20 tandem repetitive peptide sequences-19 decapeptides and 1 hexapeptide. This cDNA was expressed in yeast (9),and methods were developed to purify the recombinant adhesive protein. We also initiated studies to characterize the adhesive properties of this fragment of the mussel adhesive protein since it seemed likely to carry the basic sequences that determine moisture-resistant adhesion. In addition, we continued our genetic studies to characterize other clones encoding the mussel adhesive protein. In this study we report the isolation and characterization of a genomic clone containing the complete giant repetitive exon of the M. edulis adhesive protein, microbial expression of adhesive proteins ranging in molecular weight from 24 000 to 96 000, and demonstration of moisture-resistant adhesive properties of these proteins, which are dependent on posttranslational hydroxylation of tyrosine residues.
In turbulent intertidal zones throughout the world, marine organisms, such as mussels, attach themselves tenaciously to solid underwater surfaces (1-8). The key chemical component responsible for adhesion of the common mussel, Mytilus edulis, is believed to be a polyphenolic protein that is produced by an exocrine gland in the foot and mediates the attachment of the mussels' complex array of collagenous byssal threads to the wet solid surface. A need for the development of moisture-resistant tissue adhesives and underwater glues has recently focused research on the chemical nature of the polyphenolic protein. Amino acid composition analysis of the Mytilus edulis adhesive protein revealed a protein rich in proline, tyrosine, lysine, serine, threonine, and alanine (7). A high percentage of the proline residues are converted to 3- and 4-hydroxyproline, and the majority of tyrosine residues are hydroxylated to 3,4-dihydroxyphenylalanine(DOPA). Evidence for repeated peptide sequences in the purified polyphenolic protein was obtained by tryptic digestion Materials and Methods and amino acid composition analysis of the tryptic fragmenta (7,8). The chemically modified amino acids DOPA Cloning of M. edulis Genomic DNA. Standard methand hydroxyproline are included in the predominant trypods, essentially as described by Maniatis et al. (IO),were tic peptide Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-adapted for the purification and cloning of M . edulis Lys. Waite (7) suggested that this peptide might be genomic DNA. Mussel genomic restriction fragments of repeated up to 80 times in the mussel adhesive protein 14-18 kb from a total BglII digest were purified on lowand therefore was likely to carry key adhesive sequences. melt agarose and ligated into BamHI digested X vector In order to learn more about the structure of the adheEMBL3 (11). Gigapack Plus (Stratagene) packaging sive protein and to delineate key factors required for moisextracts were used to package the ligation mixtures into ture resistant adhesion, we have cloned DNA encoding E. coli host P2392. Screenine of the clone librarv was the majority of the mussel adhesive protein, expressed performed by hybridization to Gligonucleotideprobes GCA these sequences in yeast, and performed biochemical studAAG CCA ACT TAT AAA and A T CCT CCA ACT TAT ies with the recombinant adhesive protein. AAA, which correspond to the DNA sequence of the conWe previously reported on the cloning and microbial sensus hexapeptide sequence and the distal portion of the consensus decapeptide repeat sequence, respectively. DNA prepared from positive clones was digested * To whom correspondence and reprint requests should be with KpnI plus Hind111 and subcloned into pUC18 and addressed. 8756-7938/90/3006-0171$02.50/0
0 1990 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1990, Vol. 6,No. 3
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ThrLysHisCluProValTyrLysProValLysThrSerTyrSerAlaProTyrLysProProThrTyrClnPro ATATACACATCTTCCTTCTTAACTAACACTCTCCTTTTTTTTC~~AACAAACCATCAACCACTATACAAAC~TCTCAACACAACTTATTCCGCACCATATAAACCACCAACATACCAACCA
LeuLysLysLysValAspTyrArgProThrLysSerTyrProProThrTyrClySerLysThrAsnTyrLeuProLeuAlaLysLysLeuSerSerTyrLysProIleLysThrThrTyrAsn CTCAAAAACAAACTCCACTATCCTCCTACCAAAACTTATCCCCCAACATATGGATCAAACACAAACTATCTACCACTTGCAAAGAAGCTCTCATCTTACAAACCTATTAACACAACATATAAT
AlaLysThrAsnTyrProProVal'TyrLys ProLysMetThrTyrProProThrTyrLys ProLysProSerTyrProProThrTyrLys SerLysPro
ThrTyrLys ACATACAM
4
ProLysIleThrTyrProProThrTyrLys AlaLysProSerTyrProSerSerTyrLys ProLysLysThrTyrProProThrTyrLys ProLysLeuThrTyrProProThrTyrLys CCTAAGAAAACTTATCCCCCCACATATAAA CCTAAACTAACCTATCCTCCTACATATAAA
8
ProLysProSerTyrProProThrTyrLys ProLysProSerTyrProProSerTyrLys ThrLysLysThrTyrProSerSerTyrLys AlaLysProSerTyrProProThrTyrLys CCAAACCCCACTTATCCTCCMCATATAAA CCAAAACCAACTTATCCCCCTTCATATAAA ACTAAGAAAACTTATCCCTCTTCATATAAA GCAAAGCCAAGTTATCCTCCAACTTATAAA
12
AlaLysProSerTyrProProThrTyrLys AlaLysProSerTyrProProThrTyrLys AlaLysPro CCAAAACCAACTTATCCTCCAACTTATAAA CCAAAACCAACTTATCCTCCAACTTATAAA CCAAAGCCA
16
CCAAACACAAATTATCCACCACTTTATAAA CCTAACATCACTTATCCTCCTACATACAMCCAAAGCCCAGTTATCCTCCAACATATAAA TCAAAGCCC CCTAACATAACATACCCTCCMCATATAAA GCAAACCCAACTTATCCCTCTTCATACAAA
ThrTyrLys AlaLysProThrTyrProSerThrTyrLys ACCTATAAA CCAAAGCCAACTTATCCTTCAACGTATAAA
AlaLysProSerTyrProProThrTyrLys AlaLysPro CCIIhACCCAACTTATCCTCCAACTTATAAA CCAAACCCA
ThrTyrLys AlaLysProSerTyrProProThrTyrLys AlaLysProSerTyrProProThrTyrLys ACTTATAAA CCMCCAACTTATCCTCCAACCTATAAA GCAAAACCAACTTATCCTCCAACTTATAAA
20
AlaLysProSerTyrProProThrTyrLys AlaLysPro CCAAAACCAACTTATCCTCCAACTTATAAA GCAAACCCA
ThrTyrLys AlaLysPro ACTTATAAA GCAMGCCA
ThrTyrLys ACTTATAAA
24
AlaLysProSerTyrProProThrTyrLys AlaLysProSerTyrProProThrTyrLys AlaLysProSerTyrProProThrTyrLys AlaLysProSerTyrProProThrTyrLys GCAAACCCAACTTATCCTCCTACTTATAAA CCAAAACCCACTTATCCTCCAACTTATAAA CCAAAACCMGTTATCCTCCAACTTATAAA GCAAAACCAAGTTATCCTCCMCGTATAAA
28
AlaLysProSerTyrProProThrTyrLys AlaLysProSerTyrProProThrTyrLys AlaLysPro CCAAAACCAACTTATCCTCCAACTTATAAA CCAAAACCAACTTATCCTCCAACTTATAAA GCAAAGCCA
ThrTyrLys AlaLysProThrTyrProSerThrTyrLys ACTTATAAA GCAAAGCCCACTTATCCCTCAACGTATAAA
32
AlaLysProSerTyrProProThrTyrLys AlaLysProSerTyrProProThrTyrLys AlaLysPro CCAAACCCAACTTATCCTCCMCTTATAAA CCAAAACCAACTTATCCTCCAACTTATAAAGCAAAGCCA
ThrTyrLys AlaLysProSerTyrProPr3ThrTyrLys ACTTATAAA GCAAAGCCAACTTATCCTCCAACGTATAAA
36
AlaLysProSerTyrProProThrTyrLys AlaLysProSerTyrProProThrTyrLys AlaLysPro CCAAACCCAACTTATCCTCCAACTTATAAA CCAAAACCAACTTATCCTCCAACTTATAAA CCAAAGCCA
ThrTyrLys ALaLysProThrTyrProSerThrTyrLys ACTTATAAA GCAAACCCAACTTATCCTTCAACGTATAAA
40
AlaLysProSerTyrProProSerTyrLys AlaLysProSerTyrProProThrTyrLys AlaLysPro CCAAACCCMGTTATCCTCCATCTTATAAACCAAAACCAACTTATCCTCCAACTTATAAACCAAAGCCA
ThrTyrLys AlaLysProThrTyrProSerThrTyrLys ACTIATAM GCAAAACCAACTTATCCTTCAACGTATAAA
44
AlaLysProSerTyrProAlaSerTyrLys AlaLysProSerTyrProProThrTyrLys SerLysSerSorTyrProSerSerTyrLys P r o L y s L y s T h r T y r P r o P r o - h r T y r L y s CCAAACCCAACTTATCCACCATCTTATAAA GCAAAACCAAGCTATCCTCCAACATATAAA TCCAACTCAACTIATCCCTCTTCATACAAA CCTAACAAAACTTATCCCCCCACATATAAA
4a
ProLysLeuThrTyrLysProThrTyrLys ProLysProSerTyrProProSerTyrLys P r o L y s T h r T h r T y r P r o P r o T h r ' g r L y s ProLysIleSerTyrProProThrTyrLys CCTAAACTAACATATAAACCAACATATAAA CCAAAACCAACTTATCCACCATCTTATAAA CCTAAAACMCTTATCCTCCAACTTATAAA CCTAACATMCTTATCCTCCAACTTATAAA
52
AlaLysProSerTyrProAlaThrTyrLys AlaLysProSerTyrProProThrTyrLys AlaLysProSerTyrProProThrTyrLys AiaLysProSerTyrProProThrTyrLys CCAAAACCAACTTATCCAGCAACTTATAAA CCUCCAACTTATCCTCCAACTTACAAA GCMCCAACTTATCCTCCAACTTATAAA CCAAAACCAACTTATCCTCCAACTTATAAA
56
AlaLysPro CCAAACCCA
60
ThrTyrLys AlaLysPro ACTTATAM GCAAAGCCA
SerTyrLys AlaLysProThrTyrProSerThrTyrLys AlaLysProSerTyrProProThrTyrLys AlaLysProSerTyrProProThrTyrLys TCTTATAAA CCAAACCCAACTTATCCTTCAACGTATAAA GCAAAACCAAGTTATCCTCCAACATATAAA GCAAAACCAAGTTATCCTCCAACTTATAAA
AlaLysProSerTyrProProThrTyrLys AlaLysProThrTyrProSerThrTyrLys AlaLysProSerTyrProProThrTyrLys ProLysIleSerTyrProProThrTyrLys GCAAAACCAACTTATCCTCCAACTTATAAA GCAAAGCCAACTTACCCTTCAACCTATAAA CCAAAACCAAGTTATCCTCCAACTTATAAA CCTAAGATAAGTTATCCTCCAACTTATAAA
64
AlaLysProSerTyrProProThrTyrLys AlaLysProSerTyrProProThrTyrLys AlaLysPro CCAAAACCAACTTATCCTCCAACTTATAAA CCAAAACCAACTTATCCTCCAACTTATAAA G C M G C C A
68
ThrTyrLys AlaLysProThrAsnProSerThrTyrLys ACTTATAAA CCAAAGCCAACfMTCCTTCAACGTATAAA
AlaLysProSerTyrProProThrTyrLys AlaLysProSerTyrProProThrTvrLys AlaLysProSerTyrProProThrTyrLys AlaLysPro CCAAACCCAACTTATCCTCCAACTTATAAA CCAAAACCAACTTATCCTCCAACTTATAAA CCMCCAAGTTATCCTCCAACCTATAAA CCAAACCCA
AlaLysProThrTyrProSerThrTyrLys AlaLysPro GCAAAGCCAACTTATCCTTCAACCTATAAA CCAAACCCA
ThrTyrLys ACTTATAAA
72
ThrTyrLys AlaLysProThrTyrProProThrTyrLys AlaLysProSerTyrProProThrTyrLys ACTTATAAA CCAAACCCAACTTATCCTCCAACTTATAAA CCAAAACCAACTTATCCTCCAACATATAAA
76
ProLysProSerTyrProProThrTyrLys SerLysSerlleTyrProSerSerTyrLys ProLysLysThrTyrProProThrTyrLys ProLysLeuThrTyrProProThrTyrLys CCAAACCCAACTTATCCTCCAACTTATAAA TCCAACTCAATATATCCCTCTTCATACAAA CCTAACAAAGTTATCCCCCCACATATAAA CCTAAACTAACCTATCCTCCAACATATAAA
80
ProLysProSerTyrProProSerTyrLys ProLysIleThrTyrProSerThrTyrLys LeuLysProSerTyrProProThrTyrLys SerLysThrSerTyrProProThrTyrAsn CCAAAGCCAACTTATCCACCATCTTATAAA CCTAACATTACTTATCCCTCAACTTATAAA TTCAAGCCAAGTTATCCTCCAACATACAAA TCTAAAACAACTTACCCTCCTACATATAAC
a4
LysLysIleSerTyrProSerSerTyrLys AlaLysThrSerTyrProProAlaTyrLys PrcThrAsnArgTyr***
a6
M G A T C A C C T A T C C A T C A T C A T A T A A A CCTAACACAACTTATCCCCCAGCATATAAA CCAACAAACAGATATTM
TCTCAATATTAAAACTATTAACTAAAATATTCACATTACTCTACTACACATTTTAACGTTTCTATTCATGAGGAACACATGAACATTTGAAAGTAATACATAATCGGGCTTAATCATTTCTTA
TATTCAATCTTAATATGTTTCTCATTTCTTATCTTCTTCAACTATTCTTTCAAA~GTTTATTCTTTTCTCGTTGTGGCCTCTTATGTTTTTTCTAACACAAACTTCTATTTCTCCTATA
*
Figure 1. DNA sequence and translation product of the giant exon encoding the repetitive portion of the M.edulis adhesive protein. Underlined are intron branch and acceptor sites preceding the translated sequence. Also underlined is the polyadenylation signal AATAAA following the translated sequence. The stop codon is denoted by three asterisks, and the base preceding the poly(A) tail in cDNA clones is marked by a single asterisk.
pUC19 plasmids and M13mp19 phage. In order to have an independent DNA sequence determination of the repetitive region of the gene, DNA sequence information was obtained independently from two M13 clones with the 4.6 kb HindIII-KpnI insert and additional M13 clones with the internal 2.4 kb Sau3A insert and the 1.1 and
1.3 kb A h 1 inserts. From the procedure of Dale et al. (121, ordered sets of deletion subclones were assembled and the entire 4636 base pair DNA sequence of the HindIII-KpnI fragment was determined. Yeast Methodology. The yeast strain used in these studies was D8 (MATa leu2-3 leu2-112). Cells were trans-
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Biotechnol. Prog., 1990, Vol. 6, No. 3 BamHl
Not1
Not1 Not1
I
........... .......... ................ ................ ..........
BamHl
0 YpGX475
Not1 lest, (incomplete d& CAP
/
BamHl
Not1
\o/Not1
Not1 Not1
BamHl
LJ YpGX477
Figure 2. Genetic constructionsfor assembly of tandem repeats of the 14-1 cDNA. In a similar manner, a fourth copy of the 14-1 cDNA was inserted into YpGX477 to generate YpGX478. The stippled boxes indicate the 14-1 cDNA coding sequence. CAP is an abbreviation for calf alkaline phosphatase.
formed by the spheroplast method of Hinnen et al. (13). Synthesis of the adhesive protein was induced by overnight incubation at 30 "C in a medium composed of 1% yeast extract, 2% bactopeptone, 0.5% glucose, and 2% galactose (YPDG). The cells were broken by vortexing in the presence of glass beads, and insoluble cellular protein (which includes the adhesive protein) was pelleted by centrifugation. Expression and Purification of Streptomyces antibioticus Tyrosinase. Streptomyces antibioticus spores containing plasmid pIJ702 were received from Dr. Edward Katz (Georgetown University Medical School). Protocols for growth of Streptomyces, purification of tyrosinase, and assay of tyrosinase activity were adapted from Bernan et al. (14).
Results In the first step toward isolation of genomic clones encoding the M.edulis adhesive protein, a series of restriction digests of mussel genomic DNA was used in Southern blot hybridizations with our established polyphenolic protein cDNA and oligomer probes. The Southern blot hybridizations revealed that, for each digest, the poly-
phenolic protein repetitive sequences reside on single restriction fragments ranging in size from 2-50 kb, with one exception, AluI digestion yields two resolved fragments of approximately 1.1and 1.3 kb that hybridize to the probes. These results and low-stringencyblot experiments indicate that there is only one gene for the polyphenolic protein and that the tandem repetitive units appear to reside at a single chromosomal region of less than 3 kb. Of specific importance for isolation of the large exon encoding the adhesive protein, BglII and HindIII + KpnI digests generated adhesive protein coding fragments of 16 and 4.6 kb, respectively. A size-selected complete BglII digest of mussel DNA was used for preparation of a partial genomic library in EMBL3, and subsequent screening identified several positive clones. From one of these, a 4636 base pair HindIII-KpnI fragment was subcloned and subjected to complete DNA sequence analysis. A single huge repetitive carboxy-terminal exon was identified (Figure 1). The 1108 base pairs of upstream sequence on the Hind111 side and 606 base pairs of downstream sequence on the KpnI side that are not shown in Figure 1 are probably not coding sequences. We are able to locate the previously determined cDNA sequenceswithin the genomic sequence and find that, in some cases, cDNA slippage artifacts did occur. For example, the 14-1sequence encodes repeat units 50-56 and 74-86 found in the genomic clone. Since the sequencederived restriction map of the 4636 base pair genomic fragment agrees fully with all of our initial genomic restriction mapping described above, we have added confidence that the repetitive region of this genomic clone was not rearranged by molecular cloning. The assigned C-terminus of the protein agrees with our previous cDNA clone sequences. There are 86 tandem repeats in the large genomic exon. Seventy-two are decapeptide repeats and fourteen are hexapeptide repeats. The coding region extends upstream of the first repeat by an additional 66 amino acids. The amino acid composition of this upstream region is similar to the repeats (48 of the 66 amino acids are Tyr, Pro, Lys, Thr, and Ser), although it does not have defined repeat units. Further upstream, an intron 3' boundary exists with an excellent sequence match to the concensus yeast intron branch site sequence (ACTAAC) and an intron acceptor end (several pyrimidines (Y) followed by NYAG). The encoded polypeptide of this C-terminal exon has a molecular weight of 100 412. Posttranslational hydroxylations of the protein, as characterized by Waite's group, would increase the molecular weight of this polypeptide to about 105 000, which is still somewhat less than the estimated size of 130 000 for the polyphenolic protein (8), but the genomic sequence is in good agreement with Waite's prediction of 75-80 repetitive units in the protein. We expect that additional small exons encode the remainder of the aminoterminal region but have not yet fully characterized this region. In general, the decapeptides encoded by this genomic clone have the formula A-Lys-B-Ser/Thr-Tyr-Pro-ProThr/Ser-Tyr-Lys, where A is usually Ala or Pro and B is usually Pro. However, many sequence variations are observed. The most highly conserved residues include the tyrosines and lysines, and proline at position 6. Hexapeptide repeat units most often have the sequence Ala-Lys-Pro-Thr-Tyr-Lys. Expression of Engineered Adhesive Proteins in Yeast. Several cDNA clones encoding portions of the mussel adhesive protein were expressed in Saccharomyces cereuisiae. The 14-1 clone, which encodes the car-
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Bbtechnol. Rvg., 1990, Vol. 6, No. 3 Expression of PolymericAdhesive Proteins in Yeast
A. Genetic constructions Genetic Consttuaion
Vector
YpGX474
0-PH05 sgnal
-
"';"'
14-1 cDNA
YpGX475
YpGX477
YpGX478
B. SDS-PAGE of Yeast Strains Transformedwith Adhesive Protein Expression Vectors
Mr x kD
a b c d 200 97.4
68 43.2
10.4
Figure 3. (A) Genetic constructions for expression of genes carrying 1-4 copies of the 14-1 cDNA. (B) SDS-polyacrylamide gel electrophoresis of total insoluble yeast cell protein: (a) YpGX474; (b) YpGX475; (c) YpGX477; (d) YpGX478.
boxy terminus of the natural adhesive protein including 19 decapeptide and 1hexapeptide repeat sequences, was expressed particularly efficiently in yeast (9). The amino acid composition of the 14-1 protein is very similar to that of the entire unhydroxylated natural mussel adhesive protein, suggesting that this clone encodes an adhesive sequence representative of the intact natural protein. Therefore, studies were performed to determine if this recombinant adhesive protein has adhesive properties. The 14-1 cDNA was expressed with use of a yeast vector carrying a hybrid yeast promoter composed of segments of the S. cereuisiae GAL1 and MF-a1 promoters, a PH05 signal coding sequence, and a transcription terminator derived from a glyceraldehyde-3-phosphatedehydrogenase (GAPDH) gene (9,15).The hybrid promoter in this vector permits efficient, regulated expression of the adhesive protein. Expression is induced through the introduction of galactose as a carbon source. The PH05 signal sequence, originally included in the vector to direct secretion of the adhesive protein, functions as an effective translation initiator for expression of foreign proteins. Use of this system resulted in expression of the
adhesive protein of molecular weight of 24 0oO a t about 3-596 of the total yeast cell protein. Since molecular weight was likely to influence performance of the adhesive protein, and the natural mussel adhesive protein has a reported molecular weight of 130 OOO, the expression vector was further engineered to encode adhesive proteins of higher molecular weight. To prepare the 14-1 cDNA gene for further genetic manipulation, the cDNA was cloned into M13 and oligonucleotide-directed mutagenesis was used to insert unique restriction sites flanking the 5' and 3' sequences. As shown in Figure 2, a NotI site was positioned a t the 5' end, and the 3' end was flanked with NotI and BamHI restriction sites. The 14-1 cDNA was inserted as a NotIIBamHI fragment into YpGX283, resulting in the assembly of YpGX474. YpGX474 carries a single copy of the 14-1 cDNA. In order to generate a yeast expression vector carrying two tandem copies of the 14-1cDNA, YpGX474 was first linearized with NotI, treated with calf alkaline phosphatase, and ligated with the 14-1 cDNA fragment bordered with NotI sites a t 5' and 3' ends (Figure 2). In a similar manner, vectors carrying three (YpGX477) and four (YpGX478) tandem repeats of the 14-1 cDNA were assembled. These vectors encode adhesive proteins ranging in molecular weight from 24 OOO (YpGX474) to 96 000 (YpGX478) (see Figure 3A). Following transformation of yeast strain D8 with vectors YpGX474, YpGX475, YpGX477, and YpGX478, production of the adhesive protein was monitored by SDSpolyacrylamide gel electrophoresis and Western blot. The insoluble translation products of the recombinant yeast strains carrying one through four repeats of the 14-1cDNA sequence were then examined on SDS-polyacrylamide gels (Figure 3). The results shown in Figure 3B demonstrate that each of these proteins is expressed efficiently in yeast. The fact that the adhesive protein segregates with insoluble yeast cell protein provides a convenient first step in purification. The insoluble yeast cell paste is then suspended in a solution of 10% formic acid, resulting in selective solubilization of the adhesive protein. Following ion exchange chromatography, the adhesive protein is greater than 95% pure and can be stored as a lyophilized powder. In Vitro Hydroxylation of the Engineered Adhesive Protein. In the natural mussel adhesive protein, more than half of the tyrosine residues are posttranslationally modified to DOPA. Although the presence of DOPA residues is quite unusual in proteins, these residues have been identified in many cross-linked moistureresistant structural/adhesive proteins (16-18). Therefore, although both proline and tyrosine are posttranslationally hydroxylated in the adhesive protein, we have initially focused our studies on the contribution of modified tyrosine residues to moisture-resistant adhesivity. The adhesive protein produced in yeast does not contain DOPA residues, and these must be generated by specific in vitro hydroxylation of tyrosine residues. A tyrosinase isolated from the culture filtrate of Streptomyces antibioticus transformed with plasmid pIJ702 (14) contains both tyrosinase activity, which converts tyrosine to DOPA, and catechol oxidase activity, which converts DOPA to quinone. Using this bacterial tyrosinase, we have developed a protocol to produce a DOPA-adhesive protein in which the DOPA content represents 50% of the original tyrosine residues. The reaction solutions for adhesive protein hydroxylation contained 0.8 mg/mL adhesive protein and 25 mM
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the amount of adhesive protein bound is directly related to the initial protein concentration. Similar experiments were performed to distinguish moisture-resistant adhesion of proteins carrying DOPA or quinone residues. The results showed that the oxidation of DOPA residues is crucial for moisture-resistant adhesion. In addition, surface adhesion studies with engineered adhesive proteins ranging in molecular weight from 24 000 to 76 000 showed very similar performance. In separate experiments it was also shown that the bound adhesive protein was resistant to removal by washings with 0.9% NaCl, 0.5 7% SDS or Triton X-100, 1 % acetic acid, 0.1 N NaOH, 37 "C water, or sonication in water for 30 min.
+bacterial tyrosinase
8.0
z= 0 0
/
6.0
In
wn
a
4.0
0.00
0.50
1 .oo
AP285,
1 .so
2.00
mg/ml
Figure 4. Adhesion of tyrosinase-activated W-labeled adhesive protein was measured on Immulon I Removawells. 50-pL aliquots of adhesive protein at the indicated concentrations were added to the wells, and 5-pL of either buffer or tyrosinase (0.5 units) was added to the samples. After a 3 h incubation, the samples were removed and the wells were washed two times with water, soaked overnight with water at 37 O C , and again washed two times with water. Each well was added to a scintillation vial containing 5 mL of Aquasol, which dissolves the well. The specific activity of the protein was used to convert CPM values to milligrams protein. Each value represents the average of four wells. ascorbic acid in 0.1 M sodium phosphate, pH 7.0, and 1
unit/mL tyrosinase. This protocol is a modification of the procedures using mushroom tyrosinase described by Marumo and Waite (19). Since Streptomyces tyrosinase can convert tyrosine residues to DOPA, and DOPA to the oxidized form quinone, ascorbic acid was included to achieve a steady-state level of DOPA residues. A maximum level of hydroxylation was achieved at 1 h. The initial tyrosine concentration in the adhesive protein sample was determined from the absorbance at 275 nm to be 1.24 pmol/mL. The maximum DOPA concentration in the reaction was 0.64 pmol/mL or 52% of the initial tyrosine. Hydroxylation of each molecular weight form of the engineered adhesive protein proceeded with similar kinetics, and a similar percentage of the initial tyrosine residues were hydroxylated. The DOPA residues in the engineered adhesive protein were maintained in stable form by dialysis of the sample against 10% acetic acid, lyophilization, and storage at -20 "C.
Surface Adhesion of the Engineered Mussel Adhesive Protein. The adhesive properties of the engineered mussel adhesive proteins were tested by measuring the adherence to various surfaces in an aqueous environment. Figure 4 shows surface adhesion data from an experiment in which polystyrene was used as the substrate. In this experiment, the adhesive protein was l4Clabeled by reductive alkylation (20) and Immulon I Removawell microtiter well strips were used as a surface. Streptomyces tyrosinase (100 units/mL) was added to the adhesive protein at concentrations ranging from 0.25 to 2.0 mg/mL. In this study, no reductant was present during the reaction, and therefore quinone residues were generated. The protein solution was not allowed to dry in the wells and was removed 3 h after the addition of Streptomyces tyrosinase. After the wells were rigorously washed with water, the amount of protein bound to the surface of the wells was determined by counting the radioactivity in the individual wells. The results of this study show that the protein adheres in a moistureresistant manner, that adhesion is significantly enhanced by the in vitro modification of tyrosine residues, and that
Cross Linking of the Engineered Adhesive Protein. In order to generate a moisture-resistant adhesive with sufficient strength for various applications, it is crucial that intermolecular cross linking of polypeptide chains is achieved. It has been suggested that DOPA residues might be involved in the natural cross links formed by the mussel adhesive protein. To evaluate cross linking of this protein, we have studied two parameters. First, we used SDS-polyacrylamide gel electrophoresis to detect increased molecular weight of the protein. Second, we examined physical changes in the protein solution that would likely accompany significant increases in the protein's molecular weight. A time course of activation of the protein with a molecular weight of 24 000 with Streptomyces tyrosinase in a nonreducing environment was monitored by SDS-polyacrylamide gel electrophoresis. The gel shows the appearance of higher molecular weight bands with time and the disappearance of the 24 000 molecular weight band (Figure 5). In the 30-, 45-, and 60-min time points, discrete bands are observed with appropriate molecular weights to represent cross linking of two, three, and four molecules of the polypeptide with a molecular weight of 24 000 along with the appearance of much higher molecular weight material that does not enter the gel. In the presence of ascorbic acid, there is no evidence for cross linking over a 3-h time course. However, in the presence of ascorbic acid, DOPA residues are still generated. Therefore, these data strongly suggest that oxidation of DOPA residues is required for intermolecular cross linking. Physical changes in the protein solution concurrent with cross linking are observed for the adhesive proteins with molecular weights of 48 000 and 72 000 but not with the species with a molecular weight of 24 000. The adhesive proteins hydroxylated to carry DOPA residues were prepared as 1.3% solutions, and oxidation of the DOPA residues was catalyzed with Streptomyces tyrosinase. When 4 units/mL tyrosinase is added to the DOPA-adhesive protein in 40 mM sodium phosphate, 150 mM sodium chloride, pH 6.5, firm gels are formed with the species of molecular weights 48 000 and 72 000. The gel setting time under these conditions is about 2-3 min. The gel setting time can be controlled by the pH of the solution and the amount of enzyme. In addition, the consistency of these gels is affected by the initial protein concentration. For example, at a protein concentration of 13 mg/mL, the setting time is 2-3 min, whereas at 11 mg/mL the setting time is increased to 18-20 min. When the protein concentration was reduced to 8 mg/mL, the solution was too dilute to form a solid gel. In general, as the adhesive protein concentration is increased, the gel consistency becomes firmer. We suggest that, with the higher molecular weight forms of adhesive protein, there are increased numbers of cross links possible, resulting in polymers of very high molecular weight. In addition, the
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SDS-PAGE ANALYSIS OF THE CROSS-LINKING OF AP285
219.E 100.4
68.0 42.7
27.4
18.0 14.9
+ Figure 5. Time course of adhesive protein cross linking. The adhesive protein with a molecular weight of 24 OOO, a t 2 mg/mL in 0.1 M sodium phosphate, pH 7 , was incubated with 2 units/mL Streptomyces tyrosinase. A t the designated times, the reaction was stopped by adding 30 pL of the reaction mixture to 30 pL of 2X SDSsample buffer. The samples were analyzed on an SDS-polyacrylamide gel.
concentration of starting polypeptides will markedly affect the number of cross links in the reaction product.
Conclusions and Future Work In this paper, we have described progress in developing genetically engineered adhesive proteins based on a mussel adhesive. Our investigations have confirmed that the natural adhesive protein is composed mainly of repeated peptide sequences. In addition, the availability of genetically engineered adhesive protein has allowed us to investigate methods for achieving moistureresistant adhesion. The results of these studies demonstrate that oxidized forms of DOPA residues are involved both in the establishment of moisture-resistant surface adhesion and formation of intermolecular cross links. Further research is required to understand the chemical nature of the bonds involved in adhesion and cross linking. It has been suggested that cross links might form as a result of quinone/lysine interactions by a Michael addition reaction (2). In addition, future research is required to understand the role of hydroxyproline in the mussel adhesive. In the case of collagen, hydroxyproline residues are involved in initial alignment of polypeptide chains toward the formation of cross-linked triple helices. Since collagen is a major component of the byssal threads, it will also be interesting to probe possible interactions of the adhesive protein with collagen. Preliminary in vitro and in vivo studies have demonstrated that the engineered adhesive protein has cohesive strength in a moist environment (unpublished results). A major
goal of our research effort is to determine if these engineered adhesives can be developed into suitable materials for in vivo medical and dental applications.
Acknowledgments We thank Dr. David Anderson for purification of M. edulis genomic DNA, Jim Nagle for assistance in DNA sequence analysis, Ira Palmer, Lisa Raymond, and Anne Laws for assistance in protein chemistry, and Dr. Malcolm Finkelman and Bill Wilson for fermentations. The Genex research on bioadhesives is supported by SBIR grants from the National Institute of Dental Research.
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