Vol. 189, No. 2, 1992 December 15, 1992

MOLECULAR

BIOCHEMKAL

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CLONING

OF THE CARBOXY TERMINUS TRACHEOBRONCHIAL MUCIN’

OF A

958-964

CANINE

V. Shankar, S. Tan, M.S. Gilmore’ and G.P. Sachde?

College of Pharmacy and’College of Medicine , University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, OK 73190 Received

October

13,

1992

SUMMARY: A cDNA library constructed from canine tracheal mRNA was screened with polyclonal antiserum specific to canine tracheal apomucin (CTM-A). Eight antibody reactive clones were isolated and purified to clonality. One of the clones, designated pCIM-A, had a 1.7 kb insert and included a single open reading frame with a poly (A)+ tail. The amino acid composition of the encoded protein was consistent with that expected for CTM-A. The fusion protein produced by cloning the 1.7 kb insert in the pMALc expression vector reacted with the purified anti-apomucin CI’M-A antibody. Also, polyclonal antibodies raised to the purified protein product encoded by pCI’M-A reacted with deglycosylated CTM-A confirming that this clone does indeed code for apomucin CTM-A. This is the first report of a cDNA encoding the C-terminus of a canine tracheal mucin. 0 1992 Academic Press, Inc.

We have recently described the purification molecular weight mucins from canine tracheobronchial

and characterization

of two high

secretions (1). The major mucin

component of the mucus, namely CTM-A, had a composition typical of mucus glycoproteins with high content of hydroxy amino acids and low amounts of Cys and Met. CTM-A differed from human tracheobronchial

However,

mucins in having a higher content of aspartic

and glutamic acids. This led us to believe that the overall structure of the canine tracheal mucins may be different from that for human airway mucins. This observation was further supported by physicochemical, immunological

and molecular techniques (l-3).

‘Sequence data from this article have been deposited with the GenBank/EMBI+/DDBJ Libraries under Accession No. M3387. 2To whom correspondence 0006-291X/92

Copyright All rights

should be addressed. Fax (405) 271-3830.

$4.00

0 1992 by Academic Press, of reproduction in any form

Inc. reserved.

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Data

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Due to the high carbohydrate primary

structure

of apomucins

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

content and heterogeneity

of mucin molecules, the

cannot be successfully determined

by conventional

biochemical techniques. Using molecular techniques, cDNA clones encoding partial regions of the apomucin(s) have been obtained for human intestinal and tracheobronchial

mucins

(4-9). Employing similar approaches we have obtained a partial cDNA clone from a canine tracheal cDNA library that encodes the C-terminus of CIM-A. characterizing Furthermore,

a cDNA

encoding an identifiable

portion

This is the first report

of canine tracheal apomucin.

we show that the C-terminal region presented here for canine tracheal mucin

is unique in that it does not show any significant homology to similar regions reported for porcine submaxillary mucin (10) and human mammary tumor-associated

mucin (ll), or to

the C-terminus of mucin-like proteins from bovine submaxillary gland (12) and rat intestine

MATERIALS

AND METHODS

Purification of mucin (CTM-A), deglycosylation and production of antiserum have been described earlier (l-3). The monoclonal antibody 4F-1 (14), specific to the synthetic peptide (KYP’ITTPISTITMWPTPTPTGTQTQTPTIT) identical to the MUC2 type of repeat sequence reported in human intestinal and tracheal mucins, was kindly provided by Dr. Peter Devine, Medical Innovations Ltd., Labrador, Australia. Construction and screening of the cDNA library: Freshly removed trachea, frozen in liquid nitrogen, was used for mRNA isolation employing the PolyATtract mRNA isolation kit (Promega). The integrity of the mRNA was checked by Northern blot hybridization with a p-actin specific cDNA. The cDNA library was constructed in the UniZAP vector (Stratagene) using an oligo (dT) linker-primer to initiate first strand synthesis as per the manufacturer’s protocol. The primary library contained 2.3 x lo6 recombinants with an average insert size of 1.6 kb and was subjected to one round of amplification before screening. The cDNA library was screened using rabbit polyclonal antiserum specific to deglycosylated CTM-A using standard protocols (15). Eight antibody positive clones were obtained after screening about 8 x 16 plaques and were purified to clonality by further rounds of screening. The pBluescript (SK-) vector containing the insert was obtained by in vivo excision using helper phage R408. Three of the isolated clones had identical overlapping sequences at the 3’ end and we chose to further characterize the longest of them, designated pCTM-A (1.7 kb). Restriction

mapping and sequencing:

of pCTM-A was determined by the dideoxy and Sequenase 2.0 (U.S. Biochemicals) on When necessary dITP was substituted for ambiguities. Nested deletions were obtained

The nucleotide sequence of the 1.7 kb insert chain termination method using [%](r-dATP both single and double stranded templates. dGTP to resolve band compressions and using Exonuclease III and Sl nuclease (Erase

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-A-Base system, Promega). Sequences not obtained from nested deletions were obtained by subcloning restriction endonuclease generated fragments into pBluescript. Detection of fusion protein: The 1.7 kb insert from pCTM-A was cloned into the pMAL.c vector (New England Biolabs) downstream of the malE gene (which encodes maltose-binding protein, MBP), and expressed as a MBP fusion protein. The fusion protein was visualized upon SDS-PAGE and subsequent Western blotting using both anti-MBP antibody and polyclonal antiserum to deglycosylated CTM-A. The fusion protein was purified free of leader sequences as per the manufacturer’s protocol and the protein product specifically coded by the 1.7 kb insert was injected into rabbits to raise polyclonal antiserum. Northern blot analyses: RNA samples were subjected to electrophoresis in a 1% agarose/formaldehyde denaturing gel, transfered to GeneScreen Plus (NEN DuPont) nylon membrane and hybridized with oligonucleotide and cDNA probes under high stringency conditions using standard protocols (15). Oligonucleotide probes were 5’-end labeled to high specific activities employing [32P]Y-ATP and T4 polynucleotide kinase (Promega), and cDNA probes were labeled with [32P]a-dCTP using a random primer labeling kit (United States Biochemicals).

RESULTS

The nucleotide sequence of the 1.7 kb insert of pCTM-A

and the deduced amino

acid sequence is shown in Figure 1. This clone included a single open reading frame coding for 445 amino acid residues and an untranslated 398 bp sequence at the 3’ end preceding the poly A tail.

The composition

of the peptide encoded by the reading frame was

consistent with that expected for CTM-A.

Three potential

N-glycosylation

sites were

detected at amino acid positions 86, 207 and 345 of the open reading frame. Upon SDS-PAGE and subsequent Western blot analyses with anti-MBP antibody as well as with the polyclonal antibody to deglycosylated CTM-A, a single fusion protein band was visualized (Figure 2). The molecular weight of the fusion protein (- 97 kDa) indicated that the cDNA insert is fully expressed and encodes a protein of about 55 kDa. Polyclonal antiserum raised to the purified product encoded by the 1.7 kb cDNA insert of pCTM-A reacted with deglycosylated CTM-A in dot blot assays (data not shown) further confirming that the clone encodes a mucin type polypeptide. To further substantiate our earlier observations that the canine airway mucins may be different from that in human, we investigated the presence of the MUC2 type of repeat sequences in canine tracheal mucins.

Northern blot analysis of poly (A)+ mRNA from

canine trachea using a synthetic oligonucleotide probe specific to the conserved MUC2 type

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Am

TCG GCA CGA GCG GAT CTG TGT GTG GCT CTG GCC AAA CAC ACT ser Ala Arg Ala Asp Leu Gym Val Ala Lou Ala Lys His Thr

AGG Arg

ATG Met

ATG Met

TCT CTG AGC Ser Leu Ser

GTG GCT GAC GTC TF,! Val Ala Asp Val Phe AGC

ATC

Ser

11s

GCT Ala

ATT

CCA

GCC CGG GAC TAC Ala Arg Aep Tyr

Pro

Ile

GGT GA& MC Gly Glu As”

AGC Sez

CAC CGC TX Him

kg

TTG GAC CGC GAT GAT ATT Leu Asp Arg Asp Asp 11s

Phe

TAT AAG ATC Tyr Lys Ile

MC

Am

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GGC

ATG

TCA

CCA

GAA

Gly

llet

Sex Pro

Glu

TAC CAG Tyr Gin

CTG GAG GAG Leu Glu Glu

Tpc ATA TAT GAG GTG TCG GGC Affi Phe Ile Tyr Glu Val Ser Gly Arg

TCC AGG GAA GAC CT,! GTG CTl' ‘XT Ser Arg Glu Asp Val Val LOU Pro MC

COMMUNICATIONS

ATC

Ile

TAC CTG CGG GAG Ty+ Leu Arg Glu

121

T-3 Ser

181

CGC ACC Arg Thr

241

TCC TAC TAT GGC CTG ATG CTC TIT GGG CAC CCT CTC CTG Ser Tyr Tyr Gly Leu net. Leu Phe Gly His Pro Lou Leu

GTG TCG GTG CCC CGT GAC CGG CTC TCC TGG GAT GCC CTC TAT Ser Val Pro Arg Asp Arg Leu Ser Trp Asp Ala Leu Tyr

61

CGC kg

361

CCC AGC Pro Sar

TCG GAT GAC GAA GAT GAC GGG GAT GAG AAA GAC Ser Asp Asp Glu Asp Amp Gly Asp Glu Lys Asp

421

Val

CAC

His

ATC Ile

CTG CTG TAC Leu Leu Tyr

301

CTC TCG CGC TAT GTG ACC Le" Ser Arg Tyr Val Thr

AGA

ATA GAG GAT AAG GAC Ile Glu Asp Lys Asp

MC

ATC

CCA Pro

GGA CAC GTG GCT GGG GCC Gly His Val Ala Gly Ala

AGC Ser

CAG GAT

Ile

CCC MA Pro Lye

WC

Am

Ser

Gln

Asp

TCT

GCC Ala

CTC

TGG AGT Tq Ser

Lys

CCT Pro

GCC Ala

CCC Pro

GTG Val

541

Leu

CTG Leu

TTC

ACC

601

Ser

GGG Gly

GAT MC Asp Aen

Arg

GCC Ala

GGA Gly

TCA Ser

GGC Gly

GGG Gly

WC

CCT Pro

GGA Gly

CCA

Ser

TCT CAC Ser His

Pro

CAG Gl”

TGG CCC Trp Pro

‘XC

AGG

Arg

Arg

CGG Arg

CGG Arg

CGC

MG

CAC

Arg

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TTC MC Phe Am

Glu

CAG

AGG

GCA

Gln

Arg

Ala

CTG CAG ACA GTG AAC TCC MT Leu Gln Thr Val Asn Ser As"

GGG ACC Gly Thr

GCC

GAT TGG GAG CCA GAG ATG Asp Trp Glu Pro Glu "et

Ala

CAG Gl”

GTG GAG Val Glu

CCG TAC Pro Tyr

ATT

Ile

GCC AT’2 Ala Ile

AGC GAC CGC TCA ACC Ser Asp Arg Ser Thr MG Lys

AAG

GAG

Phe Thr

GAT ACC CAT Aap Thr His

AAG CGT TAC TAT GAC GAG Lys Arg Tyr Tyr Aep Glu

GCC GAG GGC TAC GTG ARA CAT GAC TGT GTT GGG TAC GTG CT'S AAG AAG GCT CCT Ala Glu Gly Tyr Val Lys His Asp Cys Val Gly Tyr Val Leu Lys Lya Ala Pro

GTG CGG CTG CAG GAG T'S'2 ATT GAG Val Arg Leu Gln Glu Cye Ile Glu

CTC TTC Leu Phe

ACC Thr

ACT GTC GAG ACT CTG GAG MG Thr Val Glu Thr Le" Glu Lys

GM Glu

MT As"

661 721 781

841

CCC TGG TTC TGC Pro Trp Phe Cys

CCC ACC T-32 AAG CAG Pro Thr Cys Lys Gln

CAC

AAG AAG CTG GAC CTG TGG Lye Lya Leu Asp Leu Trp

901

ATG CTG CCC GAG Met Leu Pro Glu

ACA CTC Thr Leu

CTG AAG CGC TTC TCC TAT ACC AAG TTC TCC CGC Leu Lys Arg Phe Ser Tyr Thr Lye Phe SOT Arg

961

GAG MG Glu Lye

CTG GAC ACC Asp Thr

Leu

ATC Ile

ATC Ile

CTT GTG GAG Leu Val Glu Phe

ATC AAG CCG CAG MT GAG Ile Lys Pro Gln ALI" Glu

TCC GCT Ser Ala

AAT CAT TAC GGG GGC CT-2 CGT GAC Aan His Tyyr Gly Gly Leu Arg Asp GGC Gly

CAG Gl”

TCC GAT TAC Asp Tyr

Ser

CAC His

‘lTC GAT GAC Phe Asp Asp

CCA Pro

His

CCT ATC CGG Pro Ile Arg GAG Glu

GGA CAC Gly His MC As,,

CAG CCC AGT Pro Ser

TTC ATG Phe Het

TCA

Ser

TCT GAC CCC CCG GCA Ser Asp Pro Pro Ala

GAT GTA MC Asp Val As"

TM l

GGG

GCT

GAC Asp

ACC Thr

CTG GAC Le" Asp

CTG TAC AAA TAT GAT Leu Tyr Lys Tyr Asp TAC ACA RCA Tyr Thr Thr

TIC

TCT

Phe

Ser

CTC ATC Leu Ile

GAG Glu

TIT GT-2 Phe Val

1021

GCA

GTT TCC Val Ser

1061

Ala

TIT GCT T-X Phe Ala Cys

MC

AAA GAC AGC

As”

Lys

Asp

Ser

TCA CCT GTA ACT GAG Ser Pro Val Thr Glu

MT

CAG

ATC

GAG

Asn

Gln

Ile

Glu

CGC CAG GAT GTG GCG CGT CGC CTG Arg Gln Asp Val Ala Arg Arg Leu

CM

CCC Pro

1261

TCT GAG Ser Glu

1321

CCA TCT CTG CTC

1381

AGT GTC Ser Val

TCC AAG GCA GCC TAT GTC CTC TK TAC CM Ser Lys Ala Ala Tyr Val Le" Phe Tyr Gln Gl”

CAG CTG GCC Gln Leu Ala

481

TCT CCT GCC TGC GGT TCC Ser Pro Ala Cye Gly Ser

CTG TCC Cn;

CCA

CAG

CCA Pro

GGA AGG MG

CCC

Pro

AAC A8n

Gl"

1141

1201

(445)

TG’ITCCCCGCCTGTGmGCCCCTTAGAGCA~AATC‘ITCCC~~TATTI!ATGGTl’G~CC~CCTCTGT CCTCMCCTGGGGTGTPCn;ACGCGGTGGTGGTGGGGn;C -ATCGAGACCCTGTA CCTTCTGCTGTGTATATATAAAGTGCCAGTGTGTTC~

Figure 1. The nucleotide and deduced amino acid sequence of pCTM-A. The cDNA insert of clone pCI’h&A (1.75 kb) had an open reading frame coding for 445 amino acids and an untranslated 398 bp sequence at the carboxy terminus preceding the poly (A) tail. Potential N-glycosylation sites in the sequence are underlined.

tandem repeat sequence (TTITVTPTPT, analysis of deglycosylated CTM-A

6) present in human trachea, as well as dot blot

with a monoclonal antibody specific to the MUC2 type

repeat sequence (14) showed the absence of such sequences in canine tracheal mucins (Figures 3 and 4). 961

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200k 97k 69k 46k

B

3ok

2 0 Western

ABC

Figure 2.

D

EFG

blot analysis

of MBP-pCIM-A

3 0 fusion

1

protein.

2

Lane A is pMAL

vector with no insert. Lane B is vector with pCIM-A insert and uninduced. Lane C is vector with pCTM-A insert and induced with IPTG. Lanes A-C were electrophoresed and blotted E. coli extracts probed with antibody specific to apomucin CI’M-A. Lanes D-F were duplicate samples probed with MBP antibody. The intensely stained band around 97 kDa is the MBP fusion protein. Lane G is high molecular weight rainbow protein markers (Amersham). Figure 3. Northern blot analysis of mRNA from canine and human trachea. Canine and human tracheal mRNAs (2 pg each, Lanes 1 and 2 resp.) were electrophoresed on 1% agarose-formaldehyde gel, transfered to GeneScreen Plus membrane and probed with an antisense oligonucleotide specific to MUC2 type of repeat (Panel A). After stripping the oligo probe the membrane was probed with a cDNA to p-actin to check mRNA integrity (Panel B). Arrowheads indicate positions of the 18Sand 28s ribosomal RNA. Autoradiography exposures were done at -80 “C for 24 h.

DISCUSSION In recent years there has been increasing evidence presented to suggest that, in

addition to differences in glycosylation, the heterogeneity of mucin molecules is attributable to the presence of various different polypeptide core(s) (16). A striking feature common to most mucin cDNAs cloned so far is the presence of tandemly repeating nucleotide sequences that code predominantly four mucin genes (MUCl

for hydroxy ammo acids and proline.

To date, at least

to MUC4) have been reported in humans (17,18,5,9). Of these,

the MUC2 gene product constituted at least one of the polypeptide cores of the human airway mucins with a 23 amino acid repeat feature similar to that present in human intestinal mucins while the MUC4 type represented another species with 16 amino acid tandem repeats. primarily

Recently, it has been shown that the MUCl

expressed in human mammary

gene product which is

tumors is also expressed in cultured human 962

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Figure 4. Dot blot analysis using the monoclonal antibody (4F-1) specific to MUC2 peptide sequence. Serial dilutions of the control MUC2 peptide (A), apornucin CTM-A (B) and deglycosylated human tracheobronchial mucin (C)

from 1 pg to 30 ng (l-6) were blotted on to nitrocellulose membrane and probed with the 4F-1 monoclonal antibody (culture supernatant) diluted 1:lO in phosphate buffered saline.

bronchial epithelial cells (19). These findings indicate that the mucus gel, at least in the tracheobronchial polypeptide

secretions, is comprised of complex mixture of mucins with different

cores.

Interestingly, some mucins may not contain tanclem repeats as has been shown in the cDNA encoding the polypeptide core of bovine’ submaxillary mucin (12) and two partial cDNAs encoding mucin type polypeptides in human trachea (8). Genomic organization of the MUCl

and MUC2 genes in humans has further shown that the tandem repeat regions

are situated towards the middle and the N-terminus of the mucin molecules. The C-terminal region is uniquely rich in cysteine residues and essentially devoid of any repeat motifs. The data presented in this paper for the C-terminus of a canine tracheal mucin are in agreement with earlier observations, both in terms of rather high cysteine content and the absence of any repeat motifs.

In addition, the deduced amino acid sequence of

pCTM-A includes the consensus sequence (Asn-X-Ser/Thr)

for N-glycosylation that is found

in most amino acid sequences deduced from mucin cDNAs (4,10-12).

Further, the absence

of a MUC2 type of repeat sequence in the canine trachea has been demonstrated Northern

blot experiments

with tracheal

mRNA

by both

as well as dot blot analyses of

deglycosylated mucin with the monoclonal antibody (4F-1) specific to the tandem repeat sequence in MUC2.

However, the possibility remains that repeats of different sequences

may be present in the canine tracheal mucins. Data base searches for similarity to the nucleotide and inferred amino acid sequence of the cloned canine mucin reported here revealed no significant matches.

Interestingly,

the cloned C-terminus of the canine mucin showed no significant homology to the C-terminal MUCl

sequence reported for bovine and porcine submaxillary apomucins or to the

gene product.

However, recent reports have indicated that there appears to be a 963

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lack of conservation of mucin genes among mammalian species (20,21). In view of these it would appear that canine tracheal mucins are unique among mucins characterized thus far from other mammalian

species. Work is currently in progress to isolate full length

cDNA encoding apomucin CTM-A REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

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Shankar, V., Virmani, A.K., Naziruddin, B. and Sachdev, G.P. (1991) Biochem. J. 276, 525-532. Virmani, A.K., Shankar, V., Gilmore, M.S., Graves, D.C. and Sachdev, G.P. (1991) Am. J. Respir. Cell Mol. Biol. 5, 149-154. Virmani, A.K., Naziruddin, B., Desai, V.C., Lowry, J.P., Graves, D.C. and Sachdev, G.P. (1992) In Vitro Cell. Dev. Biol. 28A, 120-127. Gum, J.R., Byrd, J.C., Hicks, J.W., Toribara, N.W., Lamport, D.T.A. and Kim, Y.S. (1989) J. Biol. Chem. 264, 6480-6487. Gum, J.R., Hicks, J.W., Swallow, D.M., Lagace, R.L., Byrd, J.C., Lamport, D.T.A., Siddiki, B. and Kim, Y.S. (1990) Biochem. Biophys. Res. Commun. 171, 407-415. Gerard, C., Eddy, R.L., Jr. and Shows, T.B. (1990) J. Clin. Invest. 86, 1921-1927. Aubert, J.P., Porchet, N., Crepin, M., Coquillaud, M.D., Vergnes, G., Mazzuca, M., Debuire,B., Petitprez,D. and Degand, P.(1991)Am. J. Respir. Cell Mol. Biol. 5,178-11 Porchet, N., Dufosse, J., Audie, J.P., Duperat, V.G., Perini, J.M., Van Cong, N., Degand, P. and Aubert, J.P. (1991) Am. Rev. Respir. Dis. 144, S15-S18. Porchet, N., Van Cong, N., Dufosse, J., Audie, J.P., Duperat, V.G., Gross, M.S., Denis, C., Bemheim, A. and Aubert, J.P. (1991) Biochem. Biophys. Res. Commun. 175, 414-422. Eckhardt, A.E., Timpte, C.S., Abernethy, J.L., Zhao, Y. and Hill, R.L. (1991) J. Biol. Chem. 266, 9678-9686. Gendler, S.J., Lancaster, C.A., Taylor-Papadimitriou, J., Duhig, T., Peat, N., Burchell, J., Pemberton, L., Lalani, E. and Wilson, D. (1990) J. Biol. Chem. 265, 15286-15293. Bhargava, A.K., Woitach, J.T., Davidson, E.A. and Bhavanandan, V.P. (1990) Proc. Natl. Acad. Sci. USA 87, 6798-6802. Xu, G., Huan, L.J., Khatri, LA., Wang, D., Bennick, A., Fahim, R.E.F., Forstner, G.G. and Forstner, J.F. (1992) J. Biol. Chem. 267, 5401-5407. Devine, P.L., McGuckin, M.A., Birrell, G.W., Whitehead, R.H., Sachdev, G.P. and Ward, B.G. (1992) Brit. J. Cancer Res. (Submitted). Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning, Vols. l&2, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Lamblin, G., Aubert, J.P., Perini, J.M., Klein, A., Porchet, N., Degand, P. and Roussel, P. (1992) Eur. Respir. J. 5, 247-256. Swallow, D.M., Gendler, S.J., Griffiths, B., Kearny, A., Povey, S., Sheer, D., Palmer, R.W. and Taylor-Papadimitriou, J. (1987) Ann. Hum. Genet. 51, 289-294. Griffiths, B., Matthews, D.J., West, L., Attwood, J., Povey, S., Swallow, D.M., Gum, J.R. and Kim, Y.S. (1990) Ann. Hum. Genet. 54, 277-285. Hollingsworth, M.A., Batra, S.K., Qi, W.N. and Yankaskas, J.R. (1992) Am. J. Respir. Cell Mol. Biol. 6, 516-520. Gum, J.R., Hicks, J.W., Lagace, R.E., Byrd, J.C., Toribara, N.W., Siddiki, B., Fearney,F.J., Lamport, D.T.A. and Kim, Y.S.(1991) J. Biol. Chem. 266, 22733-2273f Spicer,A.P., Parry,G., PattonS. and Gendler,S.J. (1991) J. Biol. Chem. 266, 15099-15105 964