Sep 3, 1987 - in bovine mucin (4). and to protect it from the external environment. Porcine. * This work was supported in part by Research Grant GM25766.
Vol 263. No. 2, Issue of January 15,pp. 1081-1088,1988 Printed in U.S. A.
OF BIOLOGICAL CHEMISTRY THEJOURNAL 0 1988 by The American Society for Biochemistry andMolecular Biology, Inc.
Porcine Submaxillary Gland Apomucin Contains Tandemly Repeated, Identical Sequences of 81 Residues* (Received for publication, September 3, 1987)
Candace S. TimpteSS, Allen E.EckhardtS, John L. Abernethyy, and Robert L. Hill+ From the Departments of $.Biochemistry and TPathology, Duke University Medical Center, Durham, North Carolina 27710
A X g t l l cDNA library, prepared from porcine sub- submaxillary mucin hasbeen examined indetail (1) and maxillary gland mRNA, was screened with anti-apo- shown to contain a polypeptide chain ( M , = 96,5001, desigmucin IgG, and five antibody-reactivephage were iso- natedapomucin,in which serine,threonine, glycine, and lated. The phage with the largestcDNA insert, desig- alanine accountfor 76% of its aminoacid composition. About nated XPSM103, was further characterized. Its fusion 70% of all the hydroxyl groups of serine and threonine are in protein reacted with anti-apomucin IgG and was used 0-glycosidic linkage with carbohydrates, ranging insize from to affinity purify antibodies that specifically reacted a monosaccharide to a pentasaccharide (2). Physical studies with apomucin, indicating that the protein shares an- suggest that apomucin is a longextendedstructurewith tigenic determinants with apomucin. The nucleotide considerable aperiodic structure and little or no secondary sequence of 1510 bases in the 3.7-kilobase cDNA insert structures that are characteristic of globular proteins (1). The ofXPSM103 has been established, thereby giving a deduced amino acid sequence of 503 residues in apo- predicted secondary structures of a 36-residue peptide from mucin, or about 45% of the molecule. The deduced porcine apomucin anda 50-residue peptide from ovine mucin sequence of the apomucin polypeptide was found to (1)support this suggestion. Mucin in solution appears to form very large aggregates contain 4.8 tandemly repeated, identical sequences of 81 residues each. The presence of these uniquely re- (M, > lo6), consistent with the view that glycosylation of peated sequences was confirmed by restriction endo- apomucin leads to the formationof long extended structures nuclease digestion ofDNA derived from XPSM103. thatinteract noncovalently. The high viscosity of mucin The repeat sequence was also confirmed in apomucin solutions results from the long, extended, hydrophilic, semiby the isolation of an 81-residue tryptic peptide with rigid structure of mucin aggregates, which are stabilized in an amino acid composition andanamino-terminal part by charge repulsion among thenegative charges on sialic amino acid sequence (up to 44 residues) identical to acid residues in the oligosaccharide prosthetic groups. those of the tandem repeat. Moreover, the peptide was In order to obtain further insight into the structural basis isolated in 760% yield, indicating that the tandem re- of the properties of mucin, it was thought desirable to deterpeat occurs at least eighttimes in apomucin. The pres- mine its amino acid sequence. This would be a formidable ence of such a long repetitive region in the gene for task by the methods of protein chemistry in view of the high apomucin raises the possibility for considerable polymorphism in the gene and a corresponding size heter- molecular weight and the rathersimple amino acid composition of apomucin. As reported here, however, the amino acid ogeneity of apomucin. The predicted secondary structure of the 503 resi- sequence can be obtained quite readily from the nucleotide dues confirms theearlier proposal that apomucin is an sequence of apomucin cDNA prepared fromsubmaxillary extended, nonglobular polypeptide. Although the se- gland mRNA. The 1510 nucleotides presented encode about molecule and surprisingly quences around 192 serine threonine and residues have 45%of theaminoacidsinthe been established in apomucin, a recognition sequence contain four tandemly repeated sequences of 243 nucleotides for the N-acetylgalactosaminyltransferasethat initi- in length. A tryptic peptide from apomucin has the identical ates glycosylation of apomucin is not evident, except sequence as that predicted by the tandemly repeated cDNA that theglycosylated residues occur in turns. sequences; and since itwas isolated in 7.6-fold molar excess, the repeated sequence likely occurs at least eight times in apomucin. Earlier sequence studies onovine apomucin (3) did not reveal any evidence of repetitive sequences within the 82 Mucin is the major glycoprotein synthesized and secreted residues that were sequenced. This is in contrast to our by Of the gland? and its findings and earlier speculations about repetitive sequences viscous aqueous sohtions serve to lubricate the oral cavity in bovine mucin (4). and to protect it from theexternalenvironment.Porcine
* This work was supported in part by Research Grant GM25766 (to R. L. H.) from the National Institute of General Medical Sciences, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotlde sequence(s)reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank withaccessionnumberfs) 503512. Supported by National Research Service Award GM07184 from the National Institute of General Medical Sciences, National Institutes of Health.
EXPERIMENTALPROCEDURES
Preparation of mRNA-Porcine submaxillary glands were removed within 2 min after death of the animal at the local abattoir. The gland (-1 g) was immediately homogenized for 30-45 s at 2-4 “C in 10 ml of 4 M guanidine isothiocyanate containing 2 M D-mercaptoethanol with a Polytron homogenizer at setting 6. The homogenates were held at 2 “C until centrifugation through a 5.7 M cesium chloride cushion for preparation of total RNA (5). The pellet was resuspended in 0.2 ml of deionized sterile water, and total RNA was precipitated from 70% ethanol. Polyadenylated RNA wasselected from total RNA by chromatography on oligo(dT)-cellulose (6). Construction and Screening of the cDNA Library-Polyadenylated
1081
1082
Submaxillary Gland Apomucin
RNA was hybridized with oligo(dT)12-15 which served as the primer dodecyl sulfate (18)and immunoblotted with anti-apomucin antibody for synthesis of the firstcDNA strand with avian myeloblastosis virus as described above. reverse transcriptase (7). Second strand synthesis was initiated by Isolation of Tryptic Peptides from Apomucin-Apomucin (3.3 mg nicking the cDNA-RNA hybrid molecules with RNase H andtreating in 6.6 ml), prepared as described earlier (l), was hydrolyzed with with DNA polymerase I and DNA ligase (8).After protection with trypsin (0.33 mg, tosylphenylalanyl chloromethyl ketone-treated, EcoRI methylase, EcoRI dodecamer linkers were ligated to thedouble- Worthington) in 0.01 M Tris. HCl at 37 'C and maintained at pH8.5 stranded cDNA, cut with EcoRI endonuclease, and ligated into the by the periodic addition of 0.1 N sodium hydroxide. Additional trypsin unique restriction site of hgtll (9). The cDNA was packaged into (0.33mg)was added after 5 h; and after an additional 4 h, the phage with an in vitro packaging extract and amplified on Escherichia hydrolysate was lyophilized, and the residue was dissolved in 10% coli strain Y1080. The library contained lo6 recombinants prior to acetic acid (2 ml). The hydrolysate (1.8 ml) was chromatographed on a column (1.8 X 145 cm) of Sephadex G-75 (fine) equilibrated and amplification. The cDNA library was screened with an IgG fraction (25 pg/ml) developed in 10% acetic acid at room temperature at a flow rate of purified from rabbit anti-apomucin antiserum (1)by the method of 30 ml/h. Fractions (3 ml) were collected, andthe peptides were Young and Davis (10) with the following modifications. EDTA (10 detected by fluorescence after autoclaving 50 pl of each fraction with mM) was included in the wash buffer, bovine serum albumin was 0.1 ml of 2 N sodium hydroxide for 30 min, neutralizing with hydrosubstituted for fetal calf serum, and 1Z51-labeledgoat anti-rabbit IgG chloric acid, and reacting with buffered o-phthalaldehyde and 0(10' cpm/filter) was the secondary antibody. Approximately 6 X lo5 mercaptoethanol (19). Fractions containing peptides werepooled, phage were screened, and five positive clones were selected and dried under vacuum, and dissolved in water. Amino Acid Compositionand Sequence Analysis of Peptides-Peppurified by subsequent rounds of rescreening. Sequencing of cDNA-Antibody-reactive, purified phage were am- tides were hydrolyzed in uacuo a t 110 "C for 24 h in constant boiling plified by the plate lysate method, and phage DNA was extracted HCl containing 0.1% phenol. After rotary evaporation, the amino (11). The cDNA inserts were excised from the phage DNA, separated acid compositions of hydrolysates were determined on a Beckman in agarose gels, and electroeluted (11). The purified inserts were 6300 high performance amino acid analyzer with buffers (lithium) provided by the manufacturer. Peptides were sequenced by automated ligated into theEcoRI site of the plasmid pEMBLmpl8 (12). Cells containing pEMBL plasmids were grown at 37 "C until the Edman degradation in an Applied Biosystems 470A Gas-Phase Seabsorbance at 600 nm was 0.3 and superinfected with phage fl (IR1) quencer. The phenylthiohydantoins weredissolved in 5% aqueous a t a multiplicity of infection of2. After 8 h, 3 ml of culture were acetonitrile containing dithiothreitol (1 mg/100 ml) and chromatosupernatant were graphed on an Applied Biosystems reverse-phase phenylthiohydancentrifuged in a Microfuge for 5 min; 2.4mlof added to 0.6 ml of 20% polyethylene glycol 6000 containing 2.5 M toin column (2.1 mm X 22 cm) in a Hewlett-Packard 1090 HPLC NaC1, mixed, and left at room temperature for 15 min. After cen- system, with the sodium acetate:tetrahydrofuran (5%) buffer (A) and trifugation for 10 min, the supernatantwas removed completely, and the acetonitrile (B) gradient recommended by the manufacturer (20). the precipitate was resuspended in 200 pl of 20 mM Tris. HC1, pH Dithiothreitol (10 mg/liter) was the reductant used in the HPLC 7.5, containing 10 mM NaCl and 0.1 mM EDTA. The templates were buffers and solvents. The amino-terminal end group of a tryptic peptide was quantitaextracted with phenolxhloroform (1:l) and then chloroform and precipitated with 70% ethanol. Pellets were washed and resuspended tively determined by "subtractive dansylation" after reaction with dansyl chloride (21). The dried peptide (50 pl, 1.2 nmol) was dissolved in 20 p1 of 10 mM Tris.HC1, pH 8.0, containing 1 mM EDTA. dryness and Nested deletion sets were created in pEMBL cDNA inserts with in 150 p1 of 0.2 M sodium bicarbonate and then taken to exonuclease 111 as described by Henikoff (13). Relative lengths of the dissolved in 150 pl of water, and 150 p1 of dansyl chloride (10 mg/ml deleted inserts were determined by gel electrophoresis of single- in acetone) were added. The reaction was allowed to proceed for 12 h at 37 "C. The reaction mixture was taken todryness and thenhydrostranded DNA sequencing templates in 1% agarose. Nucleotide sequences were determined by the dideoxy chain ter- lyzed a t 105 "C for 17 h as described above for amino acid analysis. mination method (14) using 5 pl of template, M13 template primer, The dried hydrolysate was triturated anddissolved in buffer used for amino acid analysis, and the insoluble dansylic acid was removed by and [(Y-~~SS]~ATP(YS.' Electrophoretic separations were on buffer gracentrifugation andthen chromatographed on the Beckman 6300 dient denaturing gels (E), which were then fixed with 10% acetic analyzer. acid, 10% methanol, dried, and autoradiographed 16-48 h at room Material-+" blood group-negative mucin (22), asialomucin (23), temperature. asialoafucomucin (24), and apomucin (1)were prepared as described Antibody Purification and Zmmunoblots-Antibody which specifiearlier. Asialoafucoagalactomucin was prepared by reaction of asically bound the proteins produced by the positive clone was purified aloafucomucin (10 mg) in 1 ml of 0.1 M sodium citrate, pH 6, with in the following manner. The protein from a platelysate of the phage 0.06 unit of bovine testis p-galactosidase (Boehringer-Mannheim) for was adsorbed onto a nitrocellulose filter placed on the surface of the 24 h at 37 "C. The free galactose, which accounted for 84% of the plate for 15 h. The nitrocellulose filters were incubated with 2% galactose in the starting material, was removed by dialysis. Rabbit bovine serum albumin for 1 h and then anti-apomucin antibody (25 anti-apomucin antisera and an IgG fraction were prepared as depg/ml, 16 h) andwashed as described for screening the Xgtll library, scribed earlier (1). The followingreagents were obtained commercially except that treatmentwith secondary antibody was omitted. Instead, (all enzymes were used under conditions recommended by the manantibodies absorbed to thenitrocellulose filter were eluted by washing ufacturer unless stated otherwise): PstI, SmaI, EcoRI, exonuclease three times in5 ml of 10 mM Tris. HCI, pH 7.6, containing 4M MgCl, 111, RNase H, DNA polymerase I large fragment, and M13mp18 17and 1%bovine serum albumin. The pooled washes were then dialyzed base sequencing primer (Bethesda Research Laboratories); oligo(dT)against 10 mM Tris. HCl, pH 8, containing0.15 M NaC1,l mM EDTA, cellulose, oligo(dT)lz.15,EcoRI dodecamer linkers, E. coli DNA ligase, and 0.1% Triton X-100. These antibodies were then used to detect and E. coli DNA polymerase I (Pharmacia LKB Biotechnology Inc.); purified apomucin (6 pg) after sodium dodecyl sulfate-polyacrylamide avian myeloblastosis virus reverse transcriptase (Life Sciences Assogel electrophoresis and electrophoretic transfer to nitrocellulose. The ciates); guanidine isothiocyanate (Fluka); in vitro packaging kit (Probound antibody was detected by incubation with lZSI-labeledgoat mega Biotec); nitrocellulose filters (Schleicher & Schuell); [ ( Y - ~ ~ S ] anti-rabbit IgG and autoradiography (16, 17). dATPaS (Du Pont-New England Nuclear); dithiothreitol and diThe high frequency lysogeny E. coli strain Y1089 was infected with thioerythreitol (Pierce Chemical Co.); sequenator reagents and solXPSMlO3 at a multiplicity of infection of 5. Lysogens were grown at vents (Applied Biosystems); and HPLC-grade tetrahydrofuran, meth32 "C until the absorbance at 600 nm was 0.5. Cells were then induced anol, and acetonitrile (Burdick & Jackson Laboratories Inc.). EcoRI for phage protein production by incubation at 42 "C for 20 min and methylase was a gift from Dr. Paul Modrich (Duke University Medical to a final concentra- Center) E. coli strains were Y1088 for library amplification, Y1090 addition of isopropyl-p-D-thiogalactopyranoside tion of 10 mM. Incubation was continued for an additional 1 h at for library screening, Y1089 for lysogens (lo), andNM522 for pEMBL 37 'C. The cells were centrifuged and resuspended in 10% sodium plasmid host (12). All other materials were commercial preparations dodecyl sulfate, boiled 10 min to denature proteins, and triturated of the highest quality available. through a 22-gaugeneedle to shear DNA. Bacterial and phage proteins were analyzed by polyacrylamide gel (8%) electrophoresis in sodium RESULTS The abbreviations used are: dATPaS, deoxyadenosine 5'(athi0)triphosphate [35S];HPLC, high performance liquid chromatography; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.
Characterization of the Antibody Used to Screen the cDNA Library-The reactivity of anti-apomucin IgG fraction purified from antiserum (1)was tested with mucin and its degly-
Submaxillary Gland Apomucin
1083
cosylated derivatives by immunoblottingon nitrocellulose. 12345 Only apomucin andasialoafucoagalactomucingave a positive reaction, andA blood group-negative mucin, asialomucin,and asialoafucomucin did not (data not shown). These results show that the antibody fraction reactswith protein determinants, an important consideration since the fusion protein expressed by X g t l l cDNA inserts would not be expected to be glycosylated. Construction and Screening of Submaxillary Gland cDNA -3880 bp Library-Several different methods that were used for the isolation of RNA from other tissues (25-27) gave low yields with porcine submaxillary glands. The reasons for theselow yields are unclear, but theymay have resulted from the large amount of viscous mucus in the tissue, the length of time required to remove the gland at the abattoir, or the instability -1365 bp of the RNA. Whatever the reasons for the low yields, good yields were obtained if glands were removed within 2 min of death of the animal and then immediately homogenized at -1008 bp the abattoir in guanidine isothiocyanatea t 2-4 “C. The total -720 bp RNA prepared in this manner had a 260280 nm absorbance ratio of 2. The yield of polyadenylated mRNA, which represented about 2% of the total RNA, could not be improved by scaling up the procedure. Thus, for preparation of cDNA, polyadenylated mRNA fromseveral different glandswas pooled and used to construct a cDNA library as described under “Experimental Procedures.” FIG. 1. Electrophoretic analysis of EcoRI restriction endoThe expression library containing 10‘ recombinants was nuclease digests of Xgtl 1 clones containing apomucin cDNA screened for the production of protein that was immunologi- inserts. Phage DNA was isolated from Xgtll isolates, cleaved with cally reactive with apomucin antibodies. Five antibody-reac- EcoRI restriction endonuclease, and electrophoresed on a 1% agarose tive clones were selected and plaque-purified by rescreening. gel as described under “Experimental Procedures.”Lane I , XPSMlO3; Four of these five phage isolatesrevealed two fragments upon lane 2. XPSMSOI; lane 3, hPSM802; lane 4, XPSM803; lane 5, XPSM804. The intensely staining bands at the top are XDNA. gel electrophoresis of EcoRI restriction endonuclease digests Lengths in base pairs ( b p )are indicated on the ordinate. (Fig. l),thus indicating the presenceof a n EcoRI restriction site within thegene. The largest fragment present inall four isolates was estimated tobe 2.2 kilobases; whereas the smaller nucleotides, the sequence of eachpolynucleotide was very fragments, including the fifth isolate, ranged in size from 0.7 similar and appeared to be a region encoding the same 243 to 1.5 kilobases. The phage containing the largest insert(3.7 nucleotides. Each began with a different part of the sequence, kilobases), designated XPSM103, was characterized further. and the beginnings of some were identical to the ends of Protein Produced by others.Thisresult Characterization of the Fusion suggested thattandemly repeatedseAPSM103”The proteins extracted from XPSM103-infected quences of 243nucleotidesmaybe present in the cDNA E. coli (strain Y1089) and Xgtll-infectedE. coli were separated inserts. To test this possibility, the plasmid containing the on gel electrophoresis in sodium dodecyl sulfate, transferred largest cDNA insert, pPSMlB, was digested as a function of to nitrocellulose, and immunoblotted with anti-apomucin an- time with either of two restriction endonucleases, SmaI and tibodies. An antibody-reactive protein with a lower mobility PstI, each of which was expected to cleave uniquely in the (higher molecular weight) than that of @-galactosidase was 243-nucleotide sequence. The resulting polynucleotides were found inXPSM103-infected cells, but not in Xgtll-infected or analyzed electrophoretically, as shown in Fig. 2. The partial uninfected cells (not shown). This result suggests that the digests produced polynucleotidesof sizes that were multimers fusion protein encoded by XPSM103 reacts specifically with of 243 nucleotides (243, 486, 729, and 972) and aunique apomucin antibodies. T o test, however, whether the fusion sequence joined 3’ to the multimers (435,678,921, 1164, and protein was reacting with an antibody in the antisera not 605, 848, 1091, and 1334 for SmaI). This 1407 for PstI and directed against apomucin, the anti-apomucin antiserum was suggests that there are four tandemly repeated sequences of incubated with protein extracts of XPSM103-infected cells, 243 nucleotides. Complete digests with each nuclease gave a and the antibodies specifically adsorbed were eluted. The eluted antibodies reacted with purified apomucin on immu- polynucleotide of 243 basepairs, which is the size of the repeated sequence, and apolynucleotide of the unique 3’ noblots as well as with the apomucin-&galactosidase fusion protein (not shown). This confirms that the protein encoded sequence (435 forPstI and605 for SmaI). Thisconfirms that by the cDNA insert in XPSMlO3 and apomucin share anti- the 1510-polynucleotide cDNAinsert (pPSM1B)is comprised only of the repeated sequence joined to a unique 3’-polynugenic determinants. Nucleotide Sequence of XPSM Clones-The single polynu- cleotide. The complete sequence of pPSMlB was obtained by secleotide fragment or the smaller of the two polynucleotides from EcoRI digests (Fig. 1) of each of the five phage with quence analysisof nested deletion sets constructed with exoapomucin cDNA inserts was inserted into the EcoRI endo- nuclease 111, as shown in Fig. 3. Fig. 4 shows the sequence of the 1510 nucleotides that have been sequenced on the coding nuclease site of pEMBLmpl8. The cells containing these plasmids were used to produce single-stranded phage DNA strand and the 1415 nucleotides on the antisense strand of for nucleotide sequence analysis by the dideoxy chain termi- the cDNA insert in pPSM1B. Thissequence contains a single nation method (14). Despite thesize differences in the poly- open reading frame that encodes 503 amino acids, or about
Submaxillary Gland Apomucin
1084
Sma 1
Pst 1 2 5 10 15 30 P I 2 0
2 5 10 15 30 P 120 lminl
U
kt?
.!?El
1407-1334
1164+ 972 + 921 + 729 + 678 +
I +I091
486 + 435 + 243
II
II
II
1-
PS
PS
PS
PS
P RSP FIG.2. Electrophoretic analysis of SmaI or PstI restriction endonuclease digests ofpPSMlB as a function of time. Upper, plasmid pPSMlB (10pg) was incubated with 4 units of PstI or SmaI
R
in a 40-pl final volume. Aliquots (4 pl) wereremoved at the times indicated, and the reaction was stopped by the addition of EDTA. The samples were electrophoresed on a 1.4% agarose gel, and the nucleotides were detected with ethidium bromide. P denotes pooled aliquots (1 pl) of reaction mixtures digested for 2, 5, 10, 15, and 30 min. U denotes uncut plasmid DNA. The length of the polynucleotides in base pairs (bp) is given on the right and left sides of the gel. Lower, restriction map of pPSM1B. Restriction endonuclease cleavSmaI (S),and EcoRI ( R ) . The hatched area age sites are PstI (P), represents unique sequence, the black area represents plasmid pEMBL sequence, and the open areas represent tandemly repeated sequences. 100 I
300
500
700
900
...
1300
1100
*
-
I500 pPSMlB
=
-
P
FIG.3. Nucleotide sequence strategy for exonuclease IIItreated pPSM1B. The sequence obtained from clones of exonuclease 111-deleted pPSMlB is diagramed.The arrows indicate the length of nucleotide sequence obtained and the direction of sequencing reactions.
45% of the total number (1128)of amino acid residues in apomucin (1). There aretwo features of major interest in thesequence in
?ig. 4.First, the amino acid composition of the derived amino acid sequence is contained within thecomposition of apomucin and, as listed in Table I, is particularly rich in serine, threonine, glycine, and alanine. Second, there are four tandemly repeated nucleotidesequences, 243 basesinlength (213-455,456-698,699-941, and 942-1184),that haveexactly the same sequence except a t position 1070,which was either C or A. There appear be to at least five such tandemlyrepeated sequences inview of the identityof the sequence fromnucleotides 12 to 212,except at position 99,which appears to be A, rather than C , at corresponding positions in the repeat. Fig. 5 shows the alignmentof the derived amino acid sequences of the repeated segmentsof apomucin. Isolation and Partial Sequence Analysis of a n Apomucin Tryptic Peptide-The derived amino acid sequence shown in Fig. 5 predicts thatapomucin has oneArg-Ile bond susceptible to trypsin in each of the tandemly repeated sequences. In order to confirm that derived the sequenceis thatof apomucin and to test whether apomucin contains theexpected tandemly repeated sequence, tryptic digests of apomucin were examined. Fig. 6 shows the chromatographic separation of the tryptic peptides on a column of Sephadex G-75.The elution and 390 ml. profile shows single peaks emerging at 250, 300, Based on the amino acid composition of each peak, 1.8 mg (55%) of the 2.97 mg of apomucin applied to the column emerged in the first peak and 1.0 mg (30%) in the second peak. The third peak was devoid of peptides. Remarkably, the large peak was found to contain a single peptide, designated tryptic peptide 1. Its amino acidcomposition (Table I) is identical to that of the 81-residue tandem repeat sequence shown in Fig. 5. Moreover, automated Edman degradation revealed a single amino-terminal sequence (Ile-Ser-Val-Ala-) identical to thatof the tandemly repeated sequence (Fig. 5). A reliable sequence was obtained for 38 cycles; and alanine, and 44, proline, and valine were identified at cycles 40, 42, respectively. The deduced sequence was identical for 36 residues as reported earlier (1).Quantitative end group analysis by asubtractive dansylation methodalso revealed that isoleucine decreased by46% compared with tryptic peptide1(Table I). Thedecrease in isoleucine after dansylationwould be more nearly 100% if tryptic peptide 1 contained 41 residues. This confirms that tryptic peptide 1 contains 81 residues, rather than 41 residues, as the resultof unexpected cleavage at the Arg-Pro bond. More interestingly, tryptic peptide 1 was recovered in 760% yield from the Sephadex G-75column. This yield suggests that the tandemly repeated sequenceis repeated a t least eight timesin apomucin. Prediction of the Secondary Structure of Apomucin-Fig. 7 shows the secondary structure predicted for the amino acid sequence of one tandemly repeated sequence (Fig. 5) and the unique 112 residues at the carboxyl-terminal end (Fig. 4). In accord with earlier results, the predicted secondary structure contains primarily turns with few stretches of sequence capable of forming helices or pleated sheets. DISCUSSION
A Xgtll cDNA library prepared from porcine submaxillary gland mRNA was screened with anti-apomucin antibodies, and five antibody-reactive phage were isolated on screening about 6 X lo5 phage. The nucleotide sequence of one insert containing 1510 nucleotides is reported here and shown to be comprised of four tandemly repeated, identical sequences of 243 nucleotides (Fig. 4). Although furtherstudies will be necessary to obtain thecomplete cDNA sequence of apomucin, the partial sequence reported here provides important
Submaxillary Gland Apomucin
*
*
I P a r t i a l Repeat
1085
*
*
*
*
100
G A A T T C C G G C G G A A A C T G C T A G A C C CT C T G T C G C A G G G T C A C G G A C A A C C G C A A C A G T G T G T C T G G A G C A T C A G G G T C C A C A G G A T C A T C A T C G G G A T C A A C
E
T
A
*
R
S
P
V
*
A
G
S
*
G
T
T
*
G
T
V
S
G
A
S
*
G
S
T
*
G
S
S
S
*
G
S
T
*
200
AGGCGCCACCGGAGCATCCATTGGCCAGCCCGAAACAACCAGAATCTCGGTGGCAGGCTCATCTGGAGCACCTGCAGTCTCATCTGGAGCATCACAGGCA
C
A
T
G
A
S
I
G
Q
P
E
T
S
*
Repeat 1
4 1 -
R
I
S
V
*
A
G
S
S
G
A
*
P
A
V
S
*
S
G
A
S
Q
A
*
300
GCGGGAACTTCAGCTGCTCCCCCGGGAACAACTGCCTCATCCGTCGGGGTGACGGAAACTGCTAGACCCTCTGTCGCAGGGTCAGGGACAACCGGAACAG
A
G
T
S
G
A
G
P
G
T
T
A
S
S
V
G
V
*
*
T
E
T
A
R
P
*
S
V
A
G
S
G
T
T
G
T
*
400
TCTCTGGAGCATCAGGGTCCACAGGATCATCATCGGGATCACCA~GGGCCACCGGACCATCCATTGGCCAGCCCGAAACAAGCAGAATCTCGGTGGCAGG
V
S
C
A
S
G
S
T
G
S
S
S
G
S
P
G
A
T
C
A
S
I
G
Q
P
E
T
S
R
I
S
V
A
G
*
* * * +-+Repeat 2 * * * 500 CTCATCTCGACCACCTGCAGTCTCATCTGGAGCATCACAGGCAGCGGGAACCTCAGGTGCTGGCCCGGGAACAACTGCCTCATCCGTCGGGGTGACGGAA S S C A P A V S S G A S Q A A G T S C A G P G T T A S S V G V T E *
*
*
*
*
600
ACTGCTACACCCTCTGTCGCAGGGTCAGGGACAACCGGAACAGTGTCTGGAGCATCAGGGTCCACAGGATCATCATCGGGATCACCAGGGGCCACCGGAG
T
A
R
P
S
V
A
*
G
S
G
T
T
G
T
*
V
S
G
*
A
S
G
S
T
G
S
S
S
G
*
*
S
P
G
*
A
T
G
*
T
CATCCATTCCCCAGCCCCAAACAAGCAGAATCTCGGTGGCAGGCTCATCTGGAGCACCTGCAGTCTCATCTGGAGCATCACAGGCAGCGGGAACCTCAGG
A
S
I
G
Q
P
E
T
S
R
I
S
V
A
G
S
S
G
A
P
A
V
S
S
G
A
S
Q
A
A
G
T
S
G
Repeat 3 * * * * * * * * * 800 TCCTGGCCCGGGAACAACTGCCTCATCCGTCGGGGTGACGGAAACTCCTAGACCCTCTGTCGCAGGGTCAGGGACAACCGGAACAGTGTCTGGAGCATCA A G P G T T A S G V G V T E T A R P S V A G S G T T G T V S G A S
*
*
*
*
*
*
900
GGGTCCACAGGATCATCATCGGGATCCCCAGGGGCCACCGGAGCATCCATTGGCCAGCCCGAAACAAGCAGAATCTCGGTGGCAGGCTCATCTGGAGCAC
G
S
T
G
S
S
S
G
S
P
G
A
T
G
A
S
I
G
Q
P
E
T
S
R
I
S
V
A
G
S
S
G
A
7 -
* Repeat 4 * * * 1000 CTGCAGTCTCATCTGGAGCATCACAGGCAGCGGGAACCTCAGGTGCTGGCCCGGGAACAACTGCCTCATCCGTCGGGGTGACGGAAACTGCTAGACCCTC P A V S S C A S Q A A G T S C A G P G T T A S S V G V T E T A R P S *
*
*
*
1100
TGTCGCAGGGTCAGGGACAACCGGAACAGTGTCTGGAGCATCAGGGTCCACAGGATCATCATCGGGATC-CCAGGGGCCACCGGAGCATCCATTGGCCAG V A C S G T T G T V S G A S G S T G S S S G S P G A T G A S I G Q
*
*
*
*
*
*
1200
C C C G A A A C A A G C A G A A T C T C G G T G G C A G G C T C A T C T G G A G C A C C T G C A G T C T C A T C T G G A G C A T C A C A G G C A G C G G G A A C T- T C Al G A G*G C T A C A A C T T C C A P E T S R I S V A G S S G A P A V S S G A S Q A A G T S E A T T S
*
*
*
*
*
1300
TAGAAGGTGCTGGCACTTCTGGAGTTGGATTCAAAACAGAGGCCACAACATTCCCAGGAGAAAATGAAACAACCAGAGTTGGAATCGCCACTGGTACTAC
I
E
G
A
G
T
S
G
V
G
F
K
T
E
*
A
T
T
F
P
G
E
N
E
T
T
R
V
G
I
A
T
G
T
*
*
T 1400
TGGTATAGTCTCTAGAAAGACACTGGAACCTGGAAGTTATAACACAGAGGCCACAACTTCCATAGGGAGAAGTGGGACCACCCACACAGATCTTCCAGGA
G
I
V
S
R
K
T
*
L
E
P
*
G
S
Y
N
*
T
E
A
*
T
T
S
*
I
G
R
S
G
T
T
H
T
D
*
L
P
G 1500
GCTACCACCATAGTTTTACCTGGATTCAGCCATAGTTCACAGAGTTCCAAGCCAGGCAGTTCTGTCACCACACCAGGGAGCCCAGAGTCTGGAAGTGAAA C T T I V L P G F S H S S Q S S K P C S S V T T P G S P E S G S E 1520 CAGGTACTTCACCAGAATTC T G T S G E F
FIG. 4. Nucleotide sequence of pPSMlB and deduced partial amino acid sequence of apomucin. The repetitive sequences are bracketed by arrows, and the EcoRI linker sequence used in construction of the cDNA library is underlined. The dash in the nucleotide sequence at position 1070 represents either C or A, both of which were found on sequencing this region. The one-letter code is used for the amino acids.
insights into the structure, biosynthesis, and genetic control lose-bound extract of XPSM103 reacted with apomucin on of submaxillary mucin. immunoblots. Immunological as well asstructural evidence confirm that The amino acid composition of the deduced, Partialsethe amino acid sequence deduced from the nucleotide se- quence of apomucin is in accord with that of apomucin (Table quence shown in Fig. 4 is that of apomucin. The clone I) except for lysine, leucine, phenylalanine, aspartic acid, asparagine, tyrosine, and histidine that were undetectable in XPSM103 from which the sequence was determined was isOacid hydrolysates because of their minor amounts(1-4 lated with anti-apomucin antibodies, and the fusion protein dues/mo~ecu~e) in apomucin. ~~~h are rich in serine, threeproduced by the clone reacted with anti-apomucin antibodies nine, glycine, and alanine anddevoid of certain specific amino on immunoblotting. In addition, antibodiesin anti-apomucin acids. The most conclusive structural evidence is the obserantisera that were adsorbed to and eluted from a nitrocellu- vation that thededuced sequence was identical to 44 residues
Submaxillary Gland Apomucin
1086 TABLE I
Amino acid composition of apomucin and partialsequences of aoomucin Amino acid
Partial sequence of apomucin (Fig. 4)
Apomucin
Tandemly repeated sequence (Fig. 5 )
Tryptic peptide
1
Tryptic peptide 1 after dansylation
residues/molecule
Threonine 77 11 11.0 147 9.1 Serine 294 20 115 20.7 18.9 Glutamic acid 54 20 2 4.4 3.9 Glutamine” 11 2 Proline 30 5 5.8 5.1 61 Glycine 19.8 252 105 18 19.6 Alanine 179 68 13 14.2 13.9 Valine 6 33 5.5 85 5.3 Isoleucine 15 2 0.86 28 1.6 Arginine 2 2.0 13 28 2.0 -b Lysine 3 Leucine 3 Asparagine 2 Tyrosine 1 Aspartic acid 1 Phenylalanine 4 Histidine 2 Detected as glutamic acid in acid hydrolysates. -, not detectable in acid hydrolysate becauseof small amounts relative to major amino acids. 1
I . . I l A R P S V A G S G T T G T V S G A S G S T G S S S G S T G R T G A T G A S l G ~ P f l S R l S V A G S S G A P A ~ S S G A S Q A A G l S 6E
69
311 320 330 Gly-Ala-Gly-Pro-Gly-Thr-Thr-Ala-Ser-Ser-Val-Gly-Val-Thr-Glu-Thr-Ala-Ar~-Pro-Serh
1 1
T
T t
T l
TT
Tt
b
i
t
t
T
l
t
B T
B
i
h
b
B
H
H
1
i
i
l
340 350 Val-Ala-Gly-Ser-Gly-Thr-Thr-Gly-Thr-Val-Ser-Gly-Ala-Ser-Gly-Ser-Thr-Gly-Ser-Seri~ 1 b b l i b B B B b t
t
T
T
T
T
T
i
T
T
t
T
T
T
T
T
T
T
T
T
360
370
Ser-Gly-Ala-?ro-Gly-Ala-Th~-Gly-Ala-Ser-~le-Gly-Gln-Prn-Glu-Th~-Ser-Ar~-lle-Se~h
1
T
T
T
T
T
T
i
i
i
i
i t
T
t
h
h
R
b T
T
T
t
T
t
b
i
i
h
B
B
t
380 390 Val-Ala-Gly-Ser-Ser-Gly-Ala-?ro-Ala-Val-Ser-Ser-Gly-Ala-Ser-Gln-Ala-Ala-Gly-Thr~i b b
t
l
t
r
r
r
l
i
l
~
i
h
i
i
b
r t
t
1
~
H
H
r
H
r
h
~
i
i
T
T
400
410 Ser-Glu-Ala-Thr-Thr-Ser-lle-Glu-Gly-Rla-Gly-Th~-Se~-Gly-Val-Gly-Phe-Lys-Thr-Gl~H
H
i
T
h
1
6
1
t
l
t
i
H
h
t
H
1
T
T
l
T
B
i
B
T
T
t
B
H
H
b t
w
am
w d m
A1a-Thr-~hr-Phe-?ro-G1y-Glu-Asn-Glu”Thr~-Val-Gly-lle-Ala-Thr-Gly-Th~-ThrY
h
H
i
T
T
I
T
w
w
w
w
h
B
b
1
t
1 1
h
h
B
w
w
B
B
i
B
1
B
B
1
4
w
l
b
i
B
t
T
T
l
9
440 450 Gly-lle-Val-Ser-Arg-Lys-Th~-Leu-Glu-Pro-Gly-Se~-Tyr-A~~-Th~-Gl~-Ala-Th~-lhr-Ser1 1 h H h i H H H i B B B B b b i B b l b i 1 i t i T T T T T T i t i l w w w w w w w w w w w w 470 460 Ile-Gly-Ar~-Ser-Gly-~hr-lh~-~is-Thr-Asp-te~-P~n-Gly-Gly-Thr-Th~-~le-Val-teu-Prob b i b B R l l B B B B b i T T T T T T 1 1 1 T T T l i t t
G A G Y G T i A S S V G V i I T l i R P S ~ A G S G T I G I Y S G A S G S T G S S S G S P G ~ T G ~ S l G ~ P E T S ~ ~ S Y A G S S G A P A V S S G A S ~ 1A 4A9G T S
480
490
150 G A G P G I T A S S Y G V T E T I R P S V A G S G l ~ G l V ~ G ~ S G S T G S S S G S ~ ~ A T G A S l G ~ ~ f T ~ ~ S V A G S S G A Y A ~ S S230 C A S ~ A A ~ ~ SGly-?he-Ser-His-Ser-Ser-Gln-Ser-Set-Lys-?r~-Gly-Se~-Se~-Val-Thr-Th~-Pro-Gly-Ser211
G A G P G l I A S S V G V T E T A R P S V A G S G l T G l V S G A S G S l G S S ~ G S P ~ A ~ G A S ~ G ~ ~ E T ~ ~ S V A G S S G A ~ A V S S G3A1S1 ~ A A G T S
312
G A G P G I l A S S V G ~ T E l I R P S V A G S G T l G l V S G A S G S T G S S ~ ~ S P G A l G A S l G ~ P E l S ~ S V l i G ~ S ~ A P A V S S G ~ S ~392 AAGTS
393
tATT~IEGAClSGVGFKTEATlFPGENE
...420
T T T T
T
l
T
l
T
T
T
T
t
i
i
T
T
T
T
500 Pro-Glu-Ser-Gly-Ser-Glu-Thr-Gly-Thr-Ser-Gly-Gl~-Ph~
T
T
T
T
T
T
i
T
T
T
T
FIG. 7. Predicted secondary structure (28) of the tandemly FIG. 5. Alignment of the tandemly repeated amino acid serepeated 81-residue sequence in apomucin and the unique quences (81 residues) in apomucin. The residues are numbered as described for Fig.4. The arrows indicate the Arg-Ile bond cleaved sequence carboxyl-terminal to the repeat. The letters indicate by trypsin. The Arg-Pro bonds in this sequence would not be expected the following types of secondary structure:H,strong helix former; h, weak helix former; B , strong @-sheetformer; b, weak 0-sheet former; to be cleaved. T,strong turn; t, weak turn; i, indifferent; w, aqueous; andg, hydrophobic. The residues are numberedas described for Fig. 4. 90
(Fig. 7) supports this view. This sequence, however, does not reveal definitive insight into the structural features of apo70 mucin that are recognized by the N-acetylgalactosaminyl60 transferase that incorporates N-acetylgalactosamine into gly50 cosidic linkage with the hydroxyl groups of serine and threo40 nine. Most serine and threonine residues are near glycine m 30 residues in regions with aperiodic structures. Such regions -00 20 often contain 0-linkedoligosaccharides (29). Thereis a tenda IO ency for the serine and threonine residues to be adjacent to the 30 themselves or one another. In the tandem repeat,of19 90 150 210 270 330 390 serineandthreonine residues, or about 63%, are located Volume adjacently in the sequence. A similar percentage of adjacent FIG. 6. Chromatographic separation of tryptic peptides serine and threonineresidues is also found in thesequence of (closed circles) from apomucin on Sephadex G-75. Details are apomucin that is carboxyl-terminal to the tandem repeats. It described under “Experimental Procedures.” The bar indicates the is not known, however, whether these are preferentially glyfractions pooled to give tryptic peptide 1. The open circles are the chromatographic profile of apomucin (separate experiment), which cosylated over nonadjacent hydroxyamino acid residues. It is also noteworthy that thededuced apomucin sequence was detected with anti-apomucinIgG after adsorption on nitrocellulose. is not statistically homologous with any other protein sequence registered in the GenBank. It is devoid of collagenfrom the amino terminus of a single tryptic peptide isolated like sequences (30) and shows no homology with either the proline-rich protein of mouse parotid gland (31) or the confrom apomucin (Fig. 5). Earlier studies (1)showed that apomucin isdevoid of sec- tiguous repeat polypeptide of rat submandibular glands(32). Finding four tandemly repeated, identicalsequences in the ondary structure andlikely exists in solutionas a n extended, nonglobular polypeptide that serves as a scaffold upon which apomucin cDNAinsert of XPSM103 was especially surprising. oligosaccharides are attached. The predicted secondary struc-Repetitive sequences are not uncommon and are found in ture of the deduced, partial amino acid sequence of apomucin many proteins, including Epstein-Barr virus nuclear antigen
I
80
Submaxillary Gland Apomucin (33), the a-chain of human fibrinogen (34), the low density lipoprotein receptor (35), RNA polymerase I1 (36), and chondroitin sulfate core protein (37). The tandem repetitive sequences in these other proteins, however, are much shorter than those in apomucin and are notidentical in sequence. In contrast, those in apomucin are identical in both nucleotide and amino acid sequence. Indeed, the tandem, repetitive sequences caused considerable difficulty in sequencing the cDNA in XPSMlO3 since it was difficult to know exactly which one of the tandem sequences was being sequenced. But by determining the length of the cDNA being sequenced and obtaining overlaps between two repeat sequences, 4.8 such sequences were suggested by sequence analysis (Fig. 4). This number was confirmed by the sizes of the polynucleotides obtained from restriction endonuclease digests of pPSMlB (Fig. 2). It was possible that the presence of the tandemly repeated sequences in apomucin cDNA resulted from an artifact in preparation of the cDNA library, perhaps as the result of the reverse transcriptase repeatedly transcribing a particular sequence of mRNA. This possibility was excluded, however, by isolation of a tryptic peptide from apomucin with an amino acid composition identical to 81 residues in the repeat sequence and with 38 residues from its NH2 terminus corresponding exactly to the derived sequence. Significantly, this peptide was obtained in 760% yield, consistent with the view that the 81-residue sequence is repeated at least eight times in apomucin. This number is a minimal estimate since losses of peptide undoubtedly occurred during its isolation. Knowledge of the exact number of repeats awaits furthernucleotide sequencing. If there are eight tandem repeats, they would account for 648 amino acids, which, together with the 112 amino acids (Fig. 4) carboxyl-terminal to them and not in the repeated sequence, account for 67% of the amino acids in apomucin. Likely, most of the remaining amino acids including the carboxyl-terminal region are encoded by nucleotides in the 2.2-kilobase EcoRI restriction fragment derived from XPSM103. hPSM103, however, contains only 3.7 kilobases, and its complete coding region is estimated to account for only about 85% of the apomucin polypeptide chain. cDNA encoding for the complete polypeptide, including the aminoterminal region, must be sought in other clones in order to establish the complete amino acid sequence of apomucin. Proteins other than mucin that are made by the salivary glands seem to contain many repeated sequences, although not always as identical in sequence as in mucin. Thus, the proline-rich protein from mouse parotid gland (31), the contiguous repeat protein of rat submandibular gland (32), the glue protein of Drosophila salivary gland (38), the protein products of the Balbiani rings of the midge salivary gland (39), and silk fibroin of Bombyx silk glands (40) contain repeats. The exact significance of this observation is unclear, but may indicate that these proteins with tandem repeats are structural proteins with lengths that are not constrained by the function of the protein. The genetic or evolutionary origins of the tandemly repeated, identical DNA sequences are unknown. They could arise by nonhomologous crossing over at meiosis, through the action of a transposable element or repeated transcription of a DNA sequence by polymerase. Because of the identity of the repeated sequences, however, they must have arisen very recently in porcine evolution since point mutations would accumulate if they were of ancient origin. Whatever the mechanism, the presence of the repeated sequences suggests that there could be considerable polymorphism in the apo-
1087
mucin gene giving rise to a family of apomucin polypeptides differing in length by some multiple of 81. Such hypervariability has been found in the human peanut lectin urinary mucin locus (41) that encodes for a glycoprotein with repeated sequences that arehomologous, but notidentical (42). If there is structural variability, it should be found in apomucin as well, and studies directed to thispossibility are underway. REFERENCES 1. Eckhardt, A. E., Timpte, C. S., Abernethy, J. L., Toumadje, A., Johnson, W. C., Jr., and Hill, R. L. (1987) J. Biol. Chem. 2 6 2 , 11339-11344 2. Gerken, T. A,, and Jentoft, N. (1987) Biochemistry 2 6 , 46894699 3. Hill, H. D., Jr., Schwyzer, M., Steinman, H. M., and Hill, R. L. (1977) J. Biol. Chem. 252,3799-3804 4. Pigman, W., Moschera, J., Weiss, M., and Tettamanti,G. (1973) Eur. J. Biochem. 32,148-154 5. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18,5294-5299 6. Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. U. S. A . 6 9 , 1408-1414 7. Huynh, T. V., Young, R. A., and Davis, R.W. (1985) in DNA Cloning: A Practical Approach (Glover, D. M., ed) Vol. 1, pp. 98-121, IRL, Oxford 8. Gubler, V., and Hoffman, B. J. (1983) Gene (Amst.)25,263-269 9. Young, R. A., and Davis, R.W. (1983) Proc.Natl.Acad.Sci. U. S. A. 80,1194-1198 10. Young, R. A., and Davis, R. W. (1983) Science 2 2 2 , 778-782 11. Maniatis, T., Fristch, E. F., and Sambrook, J. (1982) Molecuhr Cloning:A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, NY 12. Dente, L., Cesareni, G., and Cortese, R. (1983) Nucleic Acids Res. 11,1645-1655 13. Henikoff, S. (1984) Gene (Amst.) 2 8 , 351-359 14. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc.Natl. Acad. Sci. U. S. A . 74,5463-5467 15. Biggin, M. D., Gibson, T. J., and Hong, G. F. (1983) Proc. Natl. Acad. Sci. U. S. A . 80, 3963-3965 16. Yen, T. S. B., and Webster, R. E. (1981) J. Biol. Chem. 2 5 6 , 11259-11265 17. Burnette, W. N. (1981) Anal. Biochem. 1 1 2 , 195-203 18. Laemmli, U. K. (1970) Nature 227,680-685 19. Mendez, E., and Gavilanes, J . G. (1976) Anal. Biochem. 72,473479 20. Applied Biosystems User Bulletin No. 14 (November 18, 1985), No. 19 (May 5,1986), and No. 23 (September 24,1986), Applied Biosystems, Inc., Foster City, CA 21. Gray, W. R. (1967) Methods Enzymol. 1 1 , 139-151 22. De Salegui, M., and Plonska, H. (1969) Arch. Biochem. Biophys. 129,49-56 23 Rearick, J. I., Sadler, J. E., Paulson, J. C.. and Hill. R. L. (1979) J. Biol. Chem. 254,4444-4451 24. Beyer, T. A., Sadler, J. E., and Hill, R. L. (1980) J. Biol. Chem. 255,5364-5372 25. Favalaro, J., Freisman, R., and Kamen, R. (1980) Methods Enzymol. 6 5 , 718-749 26. Feramisco, J. R., Smart, J. E., Burridge, K., Helfman, D. M., and Thomas, G. P. (1982) J. Biol. Chem. 257,11024-11031 27. Glisin, V., Crkvenjakov, R., and Byus, C. (1974) Biochemistry 1 3 , 2633-2637 28. Chou, P. Y.,and Fasman, G. D. (1978) Annu. Reu. Biochem. 4 7 , 251-276 29. Aubert, J. P., Biserte, G., and Loucheux-Lefebvre, M. H. (1976) Arch. Biochem. Biophys. 175,410-418 30. Bornstein, P., and Traub, W. (1979) in The Proteins (Neurath, H., and Hill, R.L., eds) pp. 412-632, Academic Press, New York 31. Ann, D. K., and Carlson, D. M. (1985) J.Biol. Chem. 260,1586315872 32. Heinrich, G., and Habener, J. F. (1987) J.Biol. Chem. 262,52625270 33. Speck, S. H., Pfitzner, A., and Strominger, J. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,9298-9302 34. Doolittle, R. F., Watt, K. W. K., Cottrell, B. A., Strong, D. D., and Riley, M. (1979) Nature 2 8 0 , 464-468 '
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Submaxillary Gland Apomucin
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