Báo cáo khoa học: Mapping of the 45M1 epitope to the C-terminal cysteine-rich part of the human MUC5AC mucin potx

Mapping of the 45M1 epitope to the C-terminal cysteine-rich part of the human MUC5AC mucin Martin E. Lidell 1 , Jacques Bara 2 and Gunnar C. Hansson 1 1 Department of Medical Biochemistry, Go ¨ teborg University, Sweden 2 U-673 INSERM, Ho ˆ pital Saint-Antoine, Paris, France Mucins are large glycoproteins found on mucosal sur- faces throughout the body. They are divided into mem- brane-bound and secreted mucins, and the latter group can be further subdivided into gel-forming and non- gel-forming mucins [1]. The gel-forming mucins consti- tute the main structural component of the mucous layer protecting the underlying epithelial surfaces against the often harsh environment present in the lumen. So far, five gel-forming mucins (MUC2, MUC5B, MUC5AC, MUC6, and MUC19) have been described, and their expression appears to be tissue-specific [2–7]. In the normal situation, MUC5AC is primarily expressed in the airways and stomach, and it constitutes a major component of the respiratory and gastric mucus. The gastric M1 antigen was originally defined by several polyclonal antibodies, and it was reported that this antigen is expressed early during human colonic carcinogenesis [8,9]. In the normal gastrointestinal tract, M1 is present in the columnar mucous cells of the surface epithelium of gastric mucosa, but not in the colon [10]. However, M1 is found in the goblet cells of fetal colon [11] and in colorectal adenomas [8,9] and adenocarcinomas [10,12]. M1 has also been shown to be expressed in human pancreatic ductal adenocarcinomas [13]. Twelve hybridoma cell lines secreting mAbs against M1 have been isolated after screening of their supernatants for strong immunoreac- tivity on colon adenomas and their lack of reactivity in normal colon, as observed with the polyclonal anti- bodies against M1. Two of the mAbs, 2-11M1 and 9-13M1, have been shown to recognize epitopes present in the N-terminal cysteine-rich part of MUC5AC [14]. One mAb, 1-13M1, recognizes an epitope found in the second and fourth CysD domains of MUC5AC [14]. Another five mAbs, 19M1, 21M1, 463M, 589M, and 62M1, recognize epitopes mapped to the C-terminal cysteine-rich part of MUC5AC [15,16]. The epitopes for the last four mAbs, 2-12M1, 58M1, 166M1, and Keywords antibody; 45M1; monoclonal; MUC5AC; mucin Correspondence G. C. Hansson, Department of Medical Biochemistry, Institute of Biomedicine, Go ¨ teborg University, Box 440, 405 30 Gothenburg, Sweden Fax: +46 31 416108 Tel: +46 31 7863488 E-mail: gunnar.hansson@medkem.gu.se (Received 25 October 2007, accepted 28 November 2007) doi:10.1111/j.1742-4658.2007.06215.x Mucins are large glycoproteins protecting mucosal surfaces throughout the body. Their expressions are tissue-specific, but in disease states such as cys- tic fibrosis, inflammation and cancer, this specificity can be disturbed. MUC5AC is normally expressed in the mucous cells of the epithelia lining the stomach and the trachea, where it constitutes a major component of the gastric and respiratory mucus. A number of mAbs have been raised against the gastric M1 antigen, an early marker for colonic carcinogenesis. Several of these mAbs recognize epitopes present on MUC5AC, suggesting that MUC5AC is the antigen. However, some of the mAbs raised against the gastric M1 antigen are widely used as antibodies against MUC5AC, despite the fact that their specificity for MUC5AC has not been clearly shown. In this study, we have tested the reactivity of the latter antibodies against a recombinantly expressed C-terminal cysteine-rich part of human MUC5AC. We demonstrate for the first time that the widely used mAb 45M1, as well as 2-12M1 and 166M1, are true antibodies against MUC5AC, with epitopes located in the C-terminal cysteine-rich part of the mucin. Abbreviation GDPH, Gly-Asp-Pro-His; goat-a-mouse-AP, goat anti-(mouse IgG) coupled to alkaline phosphatase. FEBS Journal 275 (2008) 481–489 ª 2007 The Authors Journal compilation ª 2007 FEBS 481 45M1, have not been deciphered so far, although they have been tested against recombinantly expressed por- tions of MUC5AC. Despite this, 45M1 has been widely used as a mAb against MUC5AC [17–19], and is even sold as such. The 2-12M1 mAb is also sold as a mAb against MUC5AC, although its specificity for MUC5AC has not been fully established. In this study, we have tested the reactivities of the 11 mAbs towards the complete C-terminal cysteine-rich part of human MUC5AC. The results presented here show that 2-12M1, 166M1 and 45M1 really are mAbs against MUC5AC and that their epitopes are located in the C-terminal cysteine-rich part of this mucin. Results and Discussion The recombinant C-terminal cysteine-rich part of human MUC5AC In this study, the reactivities of the mAbs against M1 were tested against the C-terminal cysteine-rich part of human MUC5AC. This part was expressed in CHO-K1 cells as fusion proteins containing a myc-tag followed by either the 1041 C-terminal amino acids (M-MUC5AC- CH-long) [20] or 959 C-terminal amino acids (M-MUC5AC-CH-short) of human MUC5AC and a His-tag (Fig. 1). The murine Ig j-chain signal sequence was used to direct the protein synthesis into the secre- tory pathway. L31 [21], the cDNA clone used in previ- ous studies aimed at mapping the epitopes of the mAbs against M1 to the C-terminal part of MUC5AC [15,16], does not encode the complete C-terminal cysteine-rich part of human MUC5AC. It lacks a sequence in the 5¢-end that corresponds to the major part of the last CysD domain found in MUC5AC. The missing sequence is present in the NP3a clone reported by Meerzaman et al. [22]. The L31 and NP3a clones were used as templates when constructing the plasmids encoding M-MUC5AC-CH-long and M-MUC5AC-CH-short; the former contains the complete C-terminal cysteine- rich part, and the latter the sequence corresponding to the L31 clone with an extra nine amino acids added to its N-terminus (Fig. 1). When expressed in CHO-K1 cells, M-MUC5AC-CH-long forms disulfide-linked dimers in the endoplasmic reticulum and is partially cleaved at a Gly-Asp-Pro-His (GDPH) sequence located in the von Willebrand D4 domain during its transport through the secretory pathway [20]. After cleavage, the fragments are still held together by disulfide bonds. Reduction of these releases a C-terminal fragment (C2-H) that can be detected with a mAb against His 5 , and an N-terminal fragment (M-C1) that can be detected with a mAb against myc. Both the dimer and the monomer can be detected with either the mAb against myc or the mAb against His 5 . The recombinant MUC5AC C-terminus and its cleavage products were used to map the epitopes recognized by the mAbs against M1. Immunoreactivity of mAbs against M1 towards a recombinant MUC5AC C-terminal cysteine-rich part To test the reactivity of the mAbs against M1 towards the human MUC5AC C-terminal cysteine-rich Fig. 1. The recombinant C-terminal cysteine-rich part of human MUC5AC. A schematic picture of the recombinant C-terminal cyste- ine-rich part of human MUC5AC is given in the upper part of the figure. The GDPH-cleavage site as well as the epitopes for the mAbs against myc (amyc) and His 5 (aHis 5 ) are indicated. M-C1, N-terminal cleavage fragment; C2-H, C-terminal cleavage fragment. The lower part of the figure shows an alignment of the N-terminal MUC5AC sequences of M-MUC5AC-CH-long and M-MUC5AC-CH- short and the corresponding sequences of human, rat and mouse MUC5AC. Long, M-MUC5AC-CH long; Short, M-MUC5AC-CH short; Mouse, mouse MUC5AC (AJ511871) [29]; Rat, rat MUC5AC (U83139) [30]. ELO9 (AJ001402) [25], L31 (Z48314) [21], NP3a (U06711) [22]; GeneBank ⁄ EMBL Databank accession numbers are given in parentheses after each clone. 45M1, a human mAb against MUC5AC M. E. Lidell et al. 482 FEBS Journal 275 (2008) 481–489 ª 2007 The Authors Journal compilation ª 2007 FEBS part, cell lysate from CHO-K1 cells expressing M-MUC5AC-CH-long was separated by SDS ⁄ PAGE under both nonreducing and reducing conditions, and the proteins were blotted to membranes that were probed with the different antibodies (Fig. 2). The results after SDS ⁄ PAGE under nonreducing condi- tions (Fig. 2A) confirm previous results showing that 19M1, 21M1, 463M, 589M and 62M1 react with the C-terminal cysteine-rich part of MUC5AC [15,16]. The 1-13M1 and 2-11M1 mAbs do not react with the recombinant MUC5AC C-terminus. This is in agree- ment with previous work showing that these anti- bodies recognize epitopes in the N-terminal part of MUC5AC [14]. A triplet of reduction-sensitive bands centered around 150 kDa is detected by 1-13M1. These bands are also seen in nontransfected CHO-K1 cells, indicating that the bands are nonspecific (data not shown). More interestingly, 2-12M1, 45M1 and 166M1 react with the recombinant MUC5AC C-termi- nus. The epitopes of these antibodies have not been deciphered previously, although they have been ana- lyzed against the expression product of the L31 clone [16]. One possibility would therefore be that the epitopes of these antibodies are located within the 91 amino acids missing in the L31-encoded sequence. The 58M1 epitope does not seem to be located in the C-terminal cysteine-rich part of MUC5AC, as no staining of the recombinant protein is seen. The dimeric protein is stained by all the reacting anti- bodies. The monomeric protein is also stained, although more weakly, with all these antibodies (longer exposure times, not shown). The results after SDS ⁄ PAGE under reducing condi- tions (Fig. 2B) show that, whereas the epitopes of 2-12M1, 45M1, 463M and 589M are reduction-sensi- tive, those of 19M1 and 21M1 are not. This is in agreement with previous results [23]. In addition, the present results show that the 166M1 and 62M1 epi- topes appear to be insensitive to reduction. The fact that the recombinant MUC5AC is cleaved during its biosynthesis allows us to delimit the region where the reduction-insensitive epitopes are located. As the M-C1 fragment is detected by 19M1 and 21M1, their epitopes must be N-terminal to the GDPH-cleavage site in the von Willebrand D4 domain of MUC5AC. This is in agreement with the previously identified region between the last CysD domain and the von Willebrand D4 domain of MUC5AC [16]. The 62M1 mAb reacts with the C2-H fragment, indicating that its epitope is located C-terminally to the GDPH- cleavage site. This is also in agreement with previous studies, in which the epitope has been located to the C-terminal part of MUC5AC harboring the von Wille- brand C domain and cysteine-knot domain [16]. In the case of 166M1, a very faint band corresponding to the C2-H fragment was detected, indicating that this mAb also detects a C-terminal epitope (the band is more clearly visualized in Fig. 3D, where the blot was devel- oped for a longer time). Table 1 summarizes the reac- tivities of the antibodies towards both reduced and nonreduced M-MUC5AC-CH-long. In conclusion, our results confirm that 19M1, 21M1, 463M, 589M and 62M1 are antibodies against MUC5AC, and that their epitopes are found in the C-terminal cysteine-rich part. More importantly, we find that the previously unmapped antibodies 2-12M1, 45M1 and 166M1 also A B Fig. 2. Western blot analysis of the recombinant MUC5AC C-termi- nus using mAbs against M1. Cell lysates of CHO-K1 cells stably expressing a recombinant MUC5AC C-terminal cysteine-rich part (M-MUC5AC-CH-long) were separated by SDS ⁄ PAGE [3– 10% (w ⁄ v) gradient gel] under nonreducing (A) and reducing (B) conditions. After western blotting, the membranes were probed with mAbs against myc, His 5 or M1. The lane numbers and the mAb used for detection are indicated in the lower part of the figure. Dimer, dimeric M-MUC5AC-CH; Monomer, monomeric M-MUC5AC-CH; M-5AC-CH, reduced M-MUC5AC-CH; C2-H, C-ter- minal cleavage fragment; M-C1, N-terminal cleavage fragment. The position of the very faint band that is detected by 166M1 and that corresponds to the C2-H cleavage fragment is indicated by an asterisk in (B). Positions of molecular mass standards are indicated on the right-hand side. M. E. Lidell et al. 45M1, a human mAb against MUC5AC FEBS Journal 275 (2008) 481–489 ª 2007 The Authors Journal compilation ª 2007 FEBS 483 are antibodies against MUC5AC, with epitopes located in the MUC5AC C-terminal cysteine-rich part. Further localization of the epitopes of 45M1, 2-12 M1 and 166M1 In order to further locate the epitopes of the hitherto unmapped 45M1, 2-12M1 and 166M1, these mAbs were screened against M-MUC5AC-CH-long and M-MUC5AC-CH-short. The 45M1 mAb is sold by several companies as a specific mAb against MUC5AC, and has been used as such in a number of studies [17–19]. The specificity of 45M1 for MUC5AC has never been clearly demonstrated. As our results indicated that 45M1 reacted with a recombinant pro- tein harboring the complete C-terminal cysteine-rich part of MUC5AC, but not with a protein correspond- ing to the L31 clone, we hypothesized that the 91 amino acids located N-terminally to the latter were important for the antibody reactivity. This hypothesis was tested by using 45M1 for detection after SDS ⁄ PAGE and western blotting of cell lysates from Table 1. Immunoreactivities of mAbs against M1 towards the MUC5AC C-terminal cysteine-rich part. Staining intensity was esti- mated from ) to +++. mAbs Reduced Nonreduced M-5AC-CH M-C1 C2-H M-5AC-CH 1 Anti-myc +++ +++ ) +++ 2 1-13M1 )))) 3 2-11M1 )))) 4 2-12M1 )))+++ 5 58M1 )))) 6 19M1 ++ ++ ) +++ 7 21M1 ++ ++ ) +++ 8 45M1 )))+++ 9 166M1 + ) ++ 10 463M )))++ 11 589M )))++ 12 62M1 +++ ) +++ +++ 13 Anti-His +++ ) +++ +++ A B C D Fig. 3. Western blot analysis of the recombinant MUC5AC C-termi- nus using 45M1, 166M1 and 2-12M1. (A) Cell lysates of CHO-K1 cells and CHO-K1 cells transiently transfected with pSM-MUC5AC- CH-short were subjected to affinity purification using Dynabeads Talon beads. The purified proteins and cell lysates from CHO-K1 cells stably expressing M-MUC5AC-CH-long were analyzed by SDS ⁄ PAGE (3–10% gradient gels) under nonreducing conditions. After western blotting, the membranes were probed with either 45M1, 463M or mAb against myc (amyc). (B) Immunoprecipitations using Dynabeads coated with either 45M1 or 463M were per- formed from cell lysates of CHO-K1 cells, CHO-K1 cells transiently transfected with pSM-MUC5AC-CH-short, and CHO-K1 cells stably expressing M-MUC5AC-CH-long. The precipitates were analyzed by SDS ⁄ PAGE (3–10% gradient gels) under reducing conditions, and the proteins were blotted onto a membrane that was probed with the mAb against myc. (C) Cell lysates of CHO-K1 cells and CHO-K1 cells transiently transfected with pSM-MUC5AC-CH-short were subjected to affinity purification using Dynabeads Talon beads. The purified proteins and cell lysates from CHO-K1 cells stably express- ing M-MUC5AC-CH-long were analyzed by SDS ⁄ PAGE (3–10% gra- dient gels) under nonreducing conditions. After western blotting, the membranes were probed with 2-12M1. (D) Cell lysates from CHO-K1 cells and CHO-K1 cells stably expressing M-MUC5AC-CH- long were separated by SDS ⁄ PAGE (3–10% gel) under both reduc- ing and nonreducing conditions, and the proteins were blotted and subjected to detection by 166M1. AP, affinity purified; R, reduced samples; NR, nonreduced samples; IB, mAb used for detection; Dimer, dimeric M-MUC5AC-CH; Monomer, monomeric M-MUC5AC-CH; M-5AC-CH, reduced M-MUC5AC-CH; C2-H, C-ter- minal cleavage fragment. The weak band corresponding to the C2-H cleavage fragment is indicated by an asterisk in (D). Positions of molecular mass standards are indicated on the right-hand side. 45M1, a human mAb against MUC5AC M. E. Lidell et al. 484 FEBS Journal 275 (2008) 481–489 ª 2007 The Authors Journal compilation ª 2007 FEBS CHO-K1 cells permanently expressing M-MUC5AC- CH-long and of M-MUC5AC-CH-short affinity purified from cell lysates of CHO-K1 cells transfected with the pSM-MUC5AC-CH-short plasmid. The SDS ⁄ PAGE separation was performed under nonre- ducing conditions, as the previous results indicated that the 45M1 epitope was reduction-sensitive. The result clearly showed that the 91 amino acids N-termi- nal to the L31 expression product are crucial for 45M1 reactivity, as only M-MUC5AC-CH-long was detected (Fig. 3A). That similar amounts of M-MUC5AC-short and M-MUC5AC-long were loaded is shown by the reactivity of the mAb against myc. One reason for the requirement of the N-terminal 91 amino acids could be that these are necessary for the correct folding of the protein, and that the lack of them leads to a misfolded protein. The 463M mAb has a reduction- sensitive epitope, indicating that a correctly folded pro- tein is necessary for its reactivity. The 463M epitope has previously been mapped to the von Willebrand D4 domain of MUC5AC [16]. The result after using the 463M mAb for detection in the western blot indi- cates that M-MUC5AC-CH-short is correctly folded at least as far N-terminally as the von Willebrand D4 domain, as the reactivity of the mAb towards this protein is as strong as that against M-MUC5AC-CH- long. To rule out the possibility that the lack of reac- tivity of 45M1 against M-MUC5AC-CH-short was due to the denaturating conditions during SDS ⁄ PAGE, this mAb as well as 463M was used for immunoprecip- itations from cell lysates of cells expressing either M-MUC5AC-CH-short or M-MUC5AC-CH-long prior to SDS ⁄ PAGE and western blotting (Fig. 3B). The results after probing the membrane with the mAb against myc are consistent with those of the previ- ous experiments, as M-MUC5AC-CH-long but not M-MUC5AC-CH-short was precipitated by 45M1, whereas both forms were precipitated by 463M. Hence, the results imply that the 45M1 epitope is not destroyed during SDS ⁄ PAGE, but rather that the epi- tope is located in the part missing in M-MUC5AC- CH-short. Although one cannot exclude the possibility that the lack of the N-terminal 91 amino acids leads to an incorrectly folded protein N-terminally to the von Willebrand D4 domain, it is more likely that the epitope for 45M1 is located within this sequence. Moreover, this 91 amino acid sequence contains a part of the last CysD domain preceded by a 13 amino acid sequence that shows little homology with the corre- sponding rat and mouse sequences. In contrast, the fragment of the CysD domain, especially the sequence between amino acids 18 and 52 (in the MUC5AC sequence of M-MUC5AC-CH-long), shows strong homology between the rat, mouse and human proteins (Fig. 1). As 45M1 reacts strongly with the mucin from both rat, mouse and human [24], our results suggest that the 45M1 epitope is associated with such a sequence alone or that it is discontinuous and built up of parts found in both this sequence and more C-ter- minal ones. The 2-12M1 mAb is also sold as a mAb specific against MUC5AC, although its specificity has not been clearly demonstrated. The 2-12M1 epitope has been reported to be reduction-sensitive [12], an observation also supported by our results (Fig. 2B). The 2-12M1 mAb has previously been tested against COS-7 cells transfected with an L31-containing expression plasmid, but no reactivity against the expression product could be detected [16]. However, our initial results showed that 2-12M1 reacted with M-MUC5AC-CH-long (Fig. 2A), suggesting that the 91 amino acids that are absent in the L31 expression product could be neces- sary to build the 2-12M1 epitope. The 2-12M1 mAb was tested on western blots, like 45M1, after separa- tion of the samples by SDS ⁄ PAGE under nonreduc- ing conditions (Fig. 3C). The result shows that 2-12M1 reacted with both M-MUC5AC-long and M-MUC5AC-short. In M-MUC5AC-CH-short, an additional nine amino acids have been added N-termi- nally to the L31 expression product. One possibility is, therefore, that the epitope is located within these nine amino acids. The results definitely show that 2-12M1 is a mAb against MUC5AC and that its epitope is located in the C-terminal cysteine-rich part. Our results indicated that 166M1 detected an epitope located C-terminally to the GDPH-cleavage site, as a very faint band corresponding to the C2-H fragment was detected when probing western blots of cell lysates from CHO-K1 cells expressing M-MUC5AC-CH-long with this mAb (Fig. 2B). To rule out the possibility that the band was nonspecific, cell lysates from both CHO- K1 cells and CHO-K1 cells expressing M-MUC5AC- CH-long were separated by SDS ⁄ PAGE under reducing and nonreducing conditions, and the proteins were blotted to a membrane, which was probed with 166M1 (Fig. 3D). The results clearly show that 166M1 detects both nonreduced and reduced protein and that the epitope is located within the C-terminal cleavage fragment, as a specific band corresponding to the C2-H fragment was detected in the reduced sample. Testing 45M1, 2-12M1 and 166M1 for cross-reactivity with MUC2 The gel-forming mucins share common domain struc- tures and show a high degree of sequence homology, M. E. Lidell et al. 45M1, a human mAb against MUC5AC FEBS Journal 275 (2008) 481–489 ª 2007 The Authors Journal compilation ª 2007 FEBS 485 especially with regard to the positions of their cysteines [1]. In many cases, these mucins also present an over- lapping expression profile. Hence, it is important to determine, when a particular antibody against mucin is used, whether it shows cross-reactivity towards other mucins. Among the gel-forming mucins, the MUC5AC C-terminus shows the highest similarities with the MUC2 C-terminus. To analyze potential cross-reacti- vity, 45M1, 2-12M1 and 166M1 were tested against the C-terminal cysteine-rich domain of human MUC2 (Fig. 4). Cell lysates from CHO-K1 cells expressing either M-MUC5AC-CH-long or a recombinant MUC2 C-terminal cysteine-rich domain were separated by SDS ⁄ PAGE and blotted onto a membrane. The 45M1, 2-12 M1 and 166M1 mAbs as well as the mAb against myc were then used for detection. Only the mAb against myc detected the recombinant MUC2 C-termi- nal cysteine-rich domain, indicating that the mAbs against MUC5AC do not cross-react with MUC2. Hence, although human MUC5AC and MUC2 are highly similar, 45M1, 2-12 M1 and 166M1 seem to be specific for MUC5AC. Conclusions and future aspects This study shows that the widely used 45M1 as well as 2-12M1 and 166M1 are true mAbs against MUC5AC, with their epitopes located in the C-terminal cysteine- rich part of the protein. Fig. 5 shows a more detailed map of the epitopes for the mAbs, where 45M1 recog- nizes an epitope located in the N-terminal region of the C-terminal cysteine-rich part of MUC5AC, pre- sumably in the last CysD domain of the mucin. The epitope of 2-12M1 is located in the sequence corre- sponding to the expression product of the L31 clone, whereas the epitope for 166M1 is located C-terminally to the GDPH-cleavage site of MUC5AC. The mAbs against M1 were selected, by screening the superna- tants of hybridomas using immunohistology, as having immunoreactivity against the mucus of goblet cells of colon adenomas and a lack of reactivity against the mucus of goblet cells of normal colon [12,16,23]. It is noteworthy that among the 12 different mAbs isolated by this technique, 11 recognized the product of one unique gene: MUC5AC. The fact that the mAbs against M1 are mapped to different parts of MUC5AC Fig. 4. Testing the cross-reactivity of 45M1, 166M1 and 2-12M1 towards the C-terminal cysteine-rich domain of human MUC2. Cell lysates from CHO-K1 cells and CHO-K1 cells stably expressing either M-MUC5AC-CH-long or the corresponding C-terminal cyste- ine-rich domain of human MUC2 were separated by SDS ⁄ PAGE (3–10% gel) under nonreducing conditions. After western blotting, the mAb against myc (amyc), 45M1, 166M1 or 2-12M1 was used for detection. IB, mAb used for detection. The positions of the dimeric forms of the recombinant MUC5AC and MUC2 proteins are indicated on the left. Positions of molecular mass standards are indicated on the right-hand side. Fig. 5. Mapping of the 45M1, 166M1 and 2-12M1 epitopes on the C-terminal cysteine-rich part of human MUC5AC. The upper part of the figure shows a schematic representation of full-length human MUC5AC with its domains. The C-terminal region of the protein is expanded, and its domains and GDPH-cleavage site are indicated. In the lower part of the figure, the mapped regions for the 45M1, 166M1 and 2-12M1 epitopes are shown. 45M1, a human mAb against MUC5AC M. E. Lidell et al. 486 FEBS Journal 275 (2008) 481–489 ª 2007 The Authors Journal compilation ª 2007 FEBS makes them potentially valuable tools for future stud- ies of this protein. By use of 62M1 and the knowledge that it reacts with an epitope mapped to the von Wille- brand C domain and cysteine-knot domain of MUC5AC, it could be shown in a previous study that wild-type MUC5AC is partially cleaved at its GDPH sequence [20]. This is an example showing that the use of a well-defined mAb against M1 allows studies aimed at deciphering cleavage patterns of the full-length mucin. The present study maps the epitopes of three previously unmapped mAbs against M1 to different parts of the C-terminal cysteine-rich part of human MUC5AC. The results showing that the widely used 45M1 is a true mAb against MUC5AC are of particu- lar importance, as they verify the more than 80 previ- ous publications in which 45M1 has been used as a mAb against MUC5AC. Experimental procedures mAbs against human gastric mucin Twelve mAbs against the peptide core of gastric mucins, called mAbs against M1, were used in this study: 1-13M1, 2-11M1, 2-12M1, 9-13M1, 58M1 [12], 19M1, 21M1, 45M1 [23], 463M, 589M and 62M1 [16], and 166M1. The hybrid- oma secreting 166M1 was raised from the cell fusion used when isolating the hybridoma secreting 62M1 [16]. In short, a mouse was immunized with gastric mucin isolated from an OLe(a ) b + ) individual and purified using successive chromatography steps on Sepharose 6B and 2B. The super- natants of the hybridomas were screened, and the clones were selected by their strong reaction against the gastric surface epithelium and colon adenomas and their lack of immununoreactivity on normal colon. The mAb against myc was obtained from spent culture media of the 1-9E10.2 hybridoma (ATCC CRL-1729). Other antibodies used were: mAb against His 5 (Qiagen, Valencia, CA, USA) and goat anti-(mouse IgG) coupled to alkaline phosphatase (goat-a-mouse-AP) (Southern Biotech, Birmingham, AL, USA). Construction of MUC5AC vectors The cDNA clone L31 [21], encoding almost the complete C-terminal cysteine-rich part of human MUC5AC (lacks the N-terminal 91 amino acids of this part), inserted into a pBluescript vector, was used as template for the amplifica- tion of the MUC5AC sequence. With use of the primer pair 5¢-TATTCTAGAG AAGAGGGCCT GGTGTGCCGG AACCAGGACC AGCAGGGACC CTTCAAG-3¢ (GH262) and 5¢-ACGCGCTAGC TCAATGATGA TGATGATGGT GCATGGGGGA CACTGGGACG CC-3¢ (GH263), the MUC5AC-encoding sequence could be amplified by PCR. The primer GH262 introduced an XbaI site in the 5¢-end of the PCR product. It also added a nucleotide stretch coding for the amino acid sequence SREEGLVCR. This sequence is found N-terminally of the MUC5AC sequence encoded by the L31 sequence [22,25]. The primer GH263 introduced a His-tag and an NheI site in the 3¢-end of the PCR product. The PCR product was ligated into the XbaI site of the previ- ously described pSM vector [26]. This vector is based on the pEGFP-C1 vector (Clontech, Palo Alto, CA, USA), where the green fluorescent protein sequence has been replaced with a murine Ig j-chain signal sequence from pSec Tag A (Invitrogen, Carlsbad, CA, USA) followed by a myc-tag (EQKLISEEDL). The resulting vector, pSM-MUC5AC-CH- short, was used for transfection of CHO-K1 cells. A vector encoding the complete C-terminal cysteine-rich part, pSM-MUC5AC-CH-long, has been described before [20]. In short, NP3a [22], a cDNA clone corresponding to the 3¢-end of human MUC5AC inserted into a pBluescript vector, was used as a template for PCR. With use of the primer pair 5¢-GCTTCTAGAC ACGAGAAGAC AACCC ACTCC C-3¢ (GH287) and 5¢-GCGAGGTCTC TGTGG CGGTA TATGGTG-3¢ (GH288), the 5 ¢-part missing in the L31 clone could be amplified. GH287 introduced an XbaI site in the 5¢-end of the PCR product, and the amplified product could then be ligated into the pSM-MUC5AC- CH-short vector by its XbaI–BamHI sites. Recombinant expression and tissue culture The pSM-MUC5AC-CH-short plasmid, encoding an Ig j-chain signal sequence followed by a myc-tag, the last 959 amino acids of the human MUC5AC C-terminal cyste- ine-rich part and a His-tag, was transfected into CHO-K1 cells (ATCC CCL-61) using Lipofectamine 2000 (Invitro- gen). Twenty-four hours after transfection, cell lysates were prepared. A CHO-K1 cell line stably expressing M-MUC5AC-CH- long, the protein encoded by pSM-MUC5AC-CH-long, has been described previously [20]. This protein encodes an Ig j-chain signal sequence followed by a myc-tag, the com- plete human MUC5AC C-terminal cysteine-rich part (1041 amino acids), and a His-tag. A CHO-K1 cell line expressing the last 981 amino acids of human MUC2 (corresponding to the complete C-termi- nal cysteine-rich part) fused to an Ig j-chain signal sequence, a myc-tag and green fluorescent protein at its N-terminus has been described previously [26]. The CHO-K1 cells were cultured as described previously for LS174T [27], with the addition of 250 lgÆmL )1 G418 to the cells stably expressing recombinant proteins. Preparation of cell lysates The cell culture medium was removed, and the cells were washed twice with NaCl ⁄ P i . The cells were lysed in lysis M. E. Lidell et al. 45M1, a human mAb against MUC5AC FEBS Journal 275 (2008) 481–489 ª 2007 The Authors Journal compilation ª 2007 FEBS 487 buffer [50 mm Tris ⁄ HCl, pH 7.9, 50 mm NaCl, 1% (v ⁄ v) Triton X-100] containing protease inhibitors [2· Complete (Roche, Indianapolis, IN, USA)] and 5 mm N-ethylmalei- mide. After sonication (intensity 15) three times for 2 s (MSE Soniprep 100 sonifier), the cell debris was removed by centrifugation (16 000 g for 10 min at 4 °C). Affinity purification of recombinant MUC5AC C-terminus from cell lysates Cell lysates from CHO-K1 cells and CHO-K1 cells tran- siently transfected with the pSM-MUC5AC-CH-short plas- mid were incubated with Dynabeads Talon beads (Dynal, Oslo, Norway) for 2 h at 4 °C. The beads were washed twice with lysis buffer before the proteins were eluted in 20 mm sodium phosphate (pH 7.4), 0.5 m NaCl, and 0.2 m imidazole. Immunoprecipitation Fifty microliters of Dynabeads M-450 (Dynal) were washed three times with NaCl ⁄ P i , 0.1% BSA, and 0.1% sodium azide, and incubated on a shaker for 1 h at room tempera- ture with 30 lL of hybridoma supernatant. The beads were washed twice with lysis buffer before cell lysate was added (1 mg of total protein). After incubation overnight at 4 ° C, the beads were washed three times with lysis buffer before the proteins were released into the sample buffer with 200 mm dithiothreitol at 95 °C for 5 min. SDS ⁄ PAGE The cell lysates were mixed with Laemmli sample buffer with or without 200 mm dithiothreitol and incubated for 5 min at 95 ° C [28]. The samples were analyzed by discon- tinuous SDS ⁄ PAGE, using 3–10% gradient gels with 3% stacking gels. The molecular marker used was the Pre- cision Protein Standard (Bio-Rad, Hercules, CA, USA). Western blotting and immunodetection The proteins were transferred to poly(vinylidene difluoride) membranes (Immobilon-PSQ, 0.20 lm; Millipore) in a Transfer-Blot SD-Dry Transfer Cell (Bio-Rad) at 2.5 mAÆcm )2 for 1 h. The transfer buffer used contained 48 mm Tris, 39 mm glycine, 1.3 mm SDS, and 10% (v ⁄ v) methanol. After blotting, the membranes were placed in blocking solution and incubated overnight at 4 °C. NaCl ⁄ P i containing 5% (w ⁄ v) milk powder and 0.1% (v ⁄ v) Tween-20 was used as blocking solution when using the mAb against myc and the mAbs against M1 for detection. BSA (2%, w ⁄ v) in 10 mm Tris ⁄ HCl, 100 mm NaCl and 0.1% (v ⁄ v) Tween-20 (pH 7.5) was used as blocking solution when the mAb against His 5 was used for detection. After blocking, the membranes were incubated with either mAb against myc (1 lgÆmL )1 ), mAb against His 5 (100 ngÆmL )1 ) or the mAbs against M1 (1 : 1000) in blocking solution for 2 h at room temperature. 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Further localization of the epitopes of 45M1, 2-12 M1 and 166M1 In order to