Different Molecular Forms of TFF3 in the Human Respiratory Tract: Heterodimerization with IgG Fc Binding Protein (FCGBP) and Proteolytic Cleavage in Bronchial Secretions

The polypeptide TFF3 belongs to the trefoil factor family (TFF) of lectins. TFF3 is typically secreted from mucous epithelia together with mucins. Both intestinal and salivary TFF3 mainly exist as disulfide-linked heterodimers with IgG Fc binding protein (FCGBP). Here, we investigated bronchial tissue specimens, bronchial secretions, and bronchoalveolar lavage (BAL) fluid from patients with a chronic obstructive pulmonary disease (COPD) background by fast protein liquid chromatography and proteomics. For the first time, we identified different molecular forms of TFF3 in the lung. The high-molecular mass form represents TFF3-FCGBP oligomers, whereas the low-molecular mass forms are homodimeric and monomeric TFF3 with possibly anti-apoptotic activities. In addition, disulfide-linked TFF3 heterodimers with an Mr of about 60k and 30k were detected in both bronchial secretions and BAL fluid. In these liquids, TFF3 is partly N-terminally truncated probably by neutrophil elastase cleavage. TFF3-FCGBP is likely involved in the mucosal innate immune defense against microbial infections. We discuss a hypothetical model how TFF3 might control FCGBP oligomerization. Furthermore, we did not find indications for interactions of TFF3-FCGBP with DMBT1gp340 or the mucin MUC5AC, glycoproteins involved in mucosal innate immunity. Surprisingly, bronchial MUC5AC appeared to be degraded when compared with gastric MUC5AC.


Introduction
Human TFF3 (formerly termed hP1.B) is a secretory polypeptide consisting of 59 amino acid residues [1], which belongs to the trefoil factor family (TFF) of lectins (for reviews, see [2][3][4]). TFF3 predominantly undergoes exocrine secretion by most mucous epithelia, such as the intestine, lung, salivary glands, uterus, and vagina [1,3,[5][6][7][8][9]. Here, TFF3 is secreted together with mucins [10]. Furthermore, TFF3 is also secreted in an endocrine manner, e.g., by the hypothalamus [3,11]. TFF3 is the predominant TFF peptide in the human respiratory tract, where it is mainly synthesized by mucous acini of submucosal glands (together with the mucin MUC5B) and in varying amounts by surface goblet cells (mainly together with the mucin MUC5AC) [5]. This has been confirmed by transcriptional analysis at single-cell resolution [12]. TFF3 (together with TFF1) was also detected in the epithelium and submucosal glands of the human nasal mucosa [13] as well as in nasal polyps [14]. Of special note, the situation in the murine respiratory tract is very different, where TFF2 is the predominant TFF peptide and TFF3 is not detectable [15]. a protective function against damage by reactive oxygen species (ROS), respectively. Furthermore, we detected novel disulfide-linked TFF3 heterodimers with a relative molecular mass (Mr) of about 60k and 30k, respectively.

Characterization of TFF3 Forms in Human Bronchial Tissue Specimens
Human bronchial tissue extracts were separated by SEC and the TFF3 immunoreactivities tested ( Figure 1). Clearly, the TFF3 content peaked in a high-molecular-mass region, which also contained periodic acid-Schiff (PAS)-positive mucins, and a low-molecular-mass region ( Figure 1A). The high-molecular-mass form of TFF3 represents a disulfide-linked heterodimer with FCGBP (TFF3-FCGBP), as shown by Western blotting (Figure 1B,D). In contrast, the low-molecular-mass forms of TFF3 contain a band with an Mr of about 18k, which is probably a TFF3 homodimer [27,28], and monomeric TFF3, the latter of which is the predominant form ( Figure 1C).

Characterization of Multiple TFF3 Forms in Human Bronchial Secretions
The separation of bronchial secretions via SEC and analysis of the TFF3 forms gave a different picture when compared with bronchial tissue extracts ( Figure 2). Generally, under reducing conditions, TFF3 was always detectable as a double band, the upper band representing normal monomeric TFF3, and the lower band representing a shortened/degraded variant. A high-molecular-mass form of TFF3 peaking in fractions B8-B10 was

Characterization of Multiple TFF3 Forms in Human Bronchial Secretions
The separation of bronchial secretions via SEC and analysis of the TFF3 forms gave a different picture when compared with bronchial tissue extracts ( Figure 2). Generally, under reducing conditions, TFF3 was always detectable as a double band, the upper band representing normal monomeric TFF3, and the lower band representing a shortened/degraded variant. A high-molecular-mass form of TFF3 peaking in fractions B8-B10 was detectable in a region which also contained PAS-positive mucins (Figure 2A). This form represents a disulfide-linked heterodimer with FCGBP as shown in Figure 2B using two different anti-FCGBP antisera. This has been shown for three different bronchial secretions ( Figure 2B). detectable in a region which also contained PAS-positive mucins (Figure 2A). This form represents a disulfide-linked heterodimer with FCGBP as shown in Figure 2B using two different anti-FCGBP antisera. This has been shown for three different bronchial secretions ( Figure 2B). Furthermore, another disulfide-linked TFF3 entity was detectable mainly in fractions C2/C3, which appears with an Mr of about 60k ( Figure 2D). Additionally, a disulfidelinked TFF3 form with a Mr of about 30k was detectable in fractions around C8 ( Figure  2E). Finally, homodimeric TFF3 forms appeared in fractions D1-D5 ( Figure 2F), and monomeric TFF3 forms appeared in fractions D2-D8 ( Figure 2F). Of special note, each of the different TFF3 forms contains different amounts of the two TFF3 entities, i.e. the normal form (upper band; Figure 2A) and the degraded form (lower band; Figure 2A). For example, TFF3-FCGBP (e.g., B9) and homodimeric TFF3 (e.g., D4) mainly contain the normal TFF3 form (upper band in Figure 2A,C,F), whereas monomeric TFF3 (e.g., D6) mainly consists of the degraded form (lower band in Figure 2A,F).
In order to prove that the 60k form really contains TFF3, this species was purified from a bronchial secretion via SEC, followed by anion-exchange chromatography ( Figure  3). The 60k form accumulated in fractions C3-C10 ( Figure 3A,B). Furthermore, another disulfide-linked TFF3 entity was detectable mainly in fractions C2/C3, which appears with an M r of about 60k ( Figure 2D). Additionally, a disulfide-linked TFF3 form with a M r of about 30k was detectable in fractions around C8 ( Figure 2E). Finally, homodimeric TFF3 forms appeared in fractions D1-D5 ( Figure 2F), and monomeric TFF3 forms appeared in fractions D2-D8 ( Figure 2F). Of special note, each of the different TFF3 forms contains different amounts of the two TFF3 entities, i.e. the normal form (upper band; Figure 2A) and the degraded form (lower band; Figure 2A). For example, TFF3-FCGBP (e.g., B9) and homodimeric TFF3 (e.g., D4) mainly contain the normal TFF3 form (upper band in Figure 2A,C,F), whereas monomeric TFF3 (e.g., D6) mainly consists of the degraded form (lower band in Figure 2A,F).
In order to prove that the 60k form really contains TFF3, this species was purified from a bronchial secretion via SEC, followed by anion-exchange chromatography ( Figure 3). The 60k form accumulated in fractions C3-C10 ( Figure 3A,B). Purification of the 60k form of TFF3 from a human bronchial secretion (BS-9) and proteome analysis. (A) BS-9 was purified via SEC on a Superdex 75HL column (analogous to Figure 2) and fractions C1-C5 (60k form) were subjected to anion-exchange chromatography on a Resource Q6 column; elution profile after elution with a salt gradient (% B) as determined by absorbance at 280 nm. Underneath: Distribution of the relative TFF3 content as determined by Western blot analysis under reducing conditions and semi-quantitative analysis of the monomeric TFF3 intensities. The mucin content (PAS reaction) is shown in pink. (B) 15% SDS-PAGE under non-reducing (NR) and reducing (R) conditions, respectively, and subsequent Western blot analysis concerning TFF3 of the fractions C1-C12. Indicated is the 60k band under NR conditions. (C) 15% SDS-PAGE under NR conditions of fraction C5 (see Figure 3B), elution of the 60k-region (C5/2), and second separation of the excised band (C5/2) by 15% SDS-PAGE under NR conditions followed by Coomassie staining. Separation of the excised bands 1, 2, and 3 by 15% SDS-PAGE under reducing conditions and Western blot concerning TFF3. (D) 15% SDS-PAGE under reducing conditions of fraction C5 (see Figure  3B) followed by Coomassie staining. Separation of the excised bands 5-11 by 15% SDS-PAGE under reducing conditions and Western blotting concerning TFF3. (E) Results of the proteome analysis after tryptic in-gel digestion of the 60k-band (band 2 in Figure 3C), and bands 8 and 10 ( Figure 3D), respectively. Identified tryptic peptides belonging to TFF3 are highlighted in red.
The presence of TFF3 in the 60k-band was further demonstrated by purification via preparative non-reducing SDS-PAGE, elution of the corresponding bands 1-3 and the identification of TFF3 by Western blot analysis under reducing conditions ( Figure 3C). However, in band 2, two TFF3 forms were identified, i.e., the normal and a truncated form. Purification of the 60k form of TFF3 from a human bronchial secretion (BS-9) and proteome analysis. (A) BS-9 was purified via SEC on a Superdex 75HL column (analogous to Figure 2) and fractions C1-C5 (60k form) were subjected to anion-exchange chromatography on a Resource Q6 column; elution profile after elution with a salt gradient (% B) as determined by absorbance at 280 nm. Underneath: Distribution of the relative TFF3 content as determined by Western blot analysis under reducing conditions and semi-quantitative analysis of the monomeric TFF3 intensities. The mucin content (PAS reaction) is shown in pink. (B) 15% SDS-PAGE under non-reducing (NR) and reducing (R) conditions, respectively, and subsequent Western blot analysis concerning TFF3 of the fractions C1-C12. Indicated is the 60k band under NR conditions. (C) 15% SDS-PAGE under NR conditions of fraction C5 (see Figure 3B), elution of the 60k-region (C5/2), and second separation of the excised band (C5/2) by 15% SDS-PAGE under NR conditions followed by Coomassie staining. Separation of the excised bands 1, 2, and 3 by 15% SDS-PAGE under reducing conditions and Western blot concerning TFF3. (D) 15% SDS-PAGE under reducing conditions of fraction C5 (see Figure 3B) followed by Coomassie staining. Separation of the excised bands 5-11 by 15% SDS-PAGE under reducing conditions and Western blotting concerning TFF3. (E) Results of the proteome analysis after tryptic in-gel digestion of the 60k-band (band 2 in Figure 3C), and bands 8 and 10 ( Figure 3D), respectively. Identified tryptic peptides belonging to TFF3 are highlighted in red.
The presence of TFF3 in the 60k-band was further demonstrated by purification via preparative non-reducing SDS-PAGE, elution of the corresponding bands 1-3 and the identification of TFF3 by Western blot analysis under reducing conditions ( Figure 3C). However, in band 2, two TFF3 forms were identified, i.e., the normal and a truncated form.
In addition, TFF3 was also directly released by reduction from the 60k-band, purified via preparative SDS-PAGE and elution (bands 5-11; Figure 3D), and each band was identified by Western blot analysis ( Figure 3D). Again, two TFF3 forms were identified, i.e., a normal (band 8) and a truncated form (band 10); whereas band 9 contained both forms.
Furthermore, the purified bands with positive immunoreactivity for TFF3 (bands 2, 8, 10) were also analyzed by a proteomic approach after their tryptic digestion ( Figure 3E). Clearly, the presence of TFF3 was demonstrated in all three bands. Band 2 contained both normal TFF3 as well as a truncated form; the latter lacked four amino acid residues (i.e., EEYV) at the N-terminus. Band 8 only contained the normal TFF3 form, whereas in band 10 only the N-terminally truncated form was detectable.

Characterization of Multiple TFF3 Forms in Human BAL Fluid
As a third source for the characterization of TFF3 forms in the human lung, BAL fluid was analyzed after separation by SEC ( Figure 4). In addition, TFF3 was also directly released by reduction from the 60k-band, purified via preparative SDS-PAGE and elution (bands 5-11; Figure 3D), and each band was identified by Western blot analysis ( Figure 3D). Again, two TFF3 forms were identified, i.e., a normal (band 8) and a truncated form (band 10); whereas band 9 contained both forms.
Furthermore, the purified bands with positive immunoreactivity for TFF3 (bands 2, 8, 10) were also analyzed by a proteomic approach after their tryptic digestion ( Figure 3E). Clearly, the presence of TFF3 was demonstrated in all three bands. Band 2 contained both normal TFF3 as well as a truncated form; the latter lacked four amino acid residues (i.e., EEYV) at the N-terminus. Band 8 only contained the normal TFF3 form, whereas in band 10 only the N-terminally truncated form was detectable.

Characterization of Multiple TFF3 Forms in Human BAL Fluid
As a third source for the characterization of TFF3 forms in the human lung, BAL fluid was analyzed after separation by SEC ( Figure 4).  A high-molecular-mass form of TFF3 peaking in fractions B7/B8 represents a heterodimer with FCGBP as shown by agarose gel electrophoresis (AgGE, Figure 4B). This has also been verified from specimens originating from three different individuals using two different anti-FCGBP antisera ( Figure 4C). In contrast, all the other detectable forms were in the low-molecular-mass range ( Figure 4B).
Heterodimeric TFF3 forms are also present in the 60k-(peak at C2/C3; Figure 4E) as well as the 30k-regions (peak at C11/C12; Figure 4F,G). In the low-molecular-mass region, it is likely that homodimeric and monomeric TFF3 forms are present ( Figure 4G).
Under reducing conditions, TFF3 always appeared as a double band, i.e., an upper band representing normal monomeric TFF3, and a lower band representing a shortened/degraded form. The two TFF3 entities (normal versus shortened) appear differently in the different TFF3 forms ( Figure 4A). In particular, homodimeric TFF3 mainly contains the normal TFF3 entity ( Figure 4A); whereas monomeric TFF3 mainly consists of the shorted variant ( Figure 4A,H).
By the use of two different anti-TFF3 antisera recognizing the Cand the N-terminal portions of TFF3, it could be demonstrated that the shortened TFF3 entity (lower band) contains a truncated N-terminal region ( Figure 4H). This has been shown for both the lowand the high-molecular-mass forms, respectively ( Figure 4H).

Interactions of TFF3-FCGBP with DMBT1gp 340
In the past, homodimeric TFF3 was reported to interact in vitro with the agglutinin Deleted in Malignant Brain Tumor 1/gp340 (DMBT1gp 340 ), a glycoprotein involved in mucosal innate immunity (review: [29]). Thus, we tested whether TFF3-FCGBP and DMBT1gp 340 co-migrated after native AgGE possibly due to forming a complex ( Figure 5). For comparison, the mucin MUC5AC was also analyzed. A high-molecular-mass form of TFF3 peaking in fractions B7/B8 represents a heterodimer with FCGBP as shown by agarose gel electrophoresis (AgGE, Figure 4B). This has also been verified from specimens originating from three different individuals using two different anti-FCGBP antisera ( Figure 4C). In contrast, all the other detectable forms were in the low-molecular-mass range ( Figure 4B).
Heterodimeric TFF3 forms are also present in the 60k-(peak at C2/C3; Figure 4E) as well as the 30k-regions (peak at C11/C12; Figure 4F,G). In the low-molecular-mass region, it is likely that homodimeric and monomeric TFF3 forms are present ( Figure 4G).
Under reducing conditions, TFF3 always appeared as a double band, i.e., an upper band representing normal monomeric TFF3, and a lower band representing a shortened/degraded form. The two TFF3 entities (normal versus shortened) appear differently in the different TFF3 forms ( Figure 4A). In particular, homodimeric TFF3 mainly contains the normal TFF3 entity ( Figure 4A); whereas monomeric TFF3 mainly consists of the shorted variant ( Figure 4A,H).
By the use of two different anti-TFF3 antisera recognizing the C-and the N-terminal portions of TFF3, it could be demonstrated that the shortened TFF3 entity (lower band) contains a truncated N-terminal region ( Figure 4H). This has been shown for both the lowand the high-molecular-mass forms, respectively ( Figure 4H).

Interactions of TFF3-FCGBP with DMBT1gp 340
In the past, homodimeric TFF3 was reported to interact in vitro with the agglutinin Deleted in Malignant Brain Tumor 1/gp340 (DMBT1gp 340 ), a glycoprotein involved in mucosal innate immunity (review: [29]). Thus, we tested whether TFF3-FCGBP and DMBT1gp 340 co-migrated after native AgGE possibly due to forming a complex ( Figure 5). For comparison, the mucin MUC5AC was also analyzed.  TFF3-FCGBP was recognized as a band by both the anti-TFF3 and anti-FCGBP antisera. When compared with TFF-FCGBP, DMBT1gp 340 appeared as a smear with a lower M r , whereas bronchial MUCAC showed a smear with a somewhat higher M r . This is an indication that in the bronchial tract TFF3-FCGBP and DMBT1gp 340 are not associated. Furthermore, MUC5AC does not seem to form a complex with TFF3-FCGBP. Of special note, gastric MUC5AC appears with a much higher M r than bronchial MUC5AC.

Discussion
In the course of these studies, different molecular forms of TFF3 were detected in the respiratory tract for the first time. In human bronchial tissue extracts, a high-molecular-mass form (heterodimeric TFF3-FCGBP) as well as low-molecular-mass forms (homodimeric and predominantly monomeric TFF3) were characterized ( Figure 1). This is in agreement with results obtained from the human intestine [27] and saliva [28]. Generally, TFF3-FCGBP seems to be the predominant form of TFF3 in bronchial and intestinal tissues as well as saliva [27,28].
In contrast, in respiratory tract secretions (bronchial secretions and BAL fluid, respectively) additional TFF3 forms were detected, i.e., the heterodimeric 60k-and 30k-forms, respectively (Figures 2 and 4). Additionally, little homodimeric TFF3 and mainly monomeric TFF3 represented the low-molecular-mass forms of TFF3 (Figures 2 and 4). Furthermore, in the secretory specimens, TFF3 appeared as two entities, a normal TFF3 (upper band) and a degraded variant (lower band) lacking the four N-terminal amino acid residues (Figures 3  and 4). Of note, and in contrast to the bronchial tissue extracts, in the secretory material the TFF3-FCGBP heterodimer does not represent the predominant TFF3 form anymore (Figures 2 and 4).
The results were obtained from specimens from patients with a COPD background, which is characterized by an abnormal inflammatory reaction. This might be a limit of this study as both TFF3 and FCGBP are up-regulated during inflammation, e.g., by interleukin-13 (IL-13). However, both TFF3 and FCGBP are also typically expressed in the mucous cells of submucosal glands of normal control lung tissue [12,30]. Thus, the presence of the TFF3-FCGBP heterodimers in healthy lung tissue can be anticipated.

TFF3 from the Human Respiratory Tract Forms High-Molecular-Mass Heterodimers with FCGBP
Currently, the biological role of TFF3-FCGBP and FCGBP, respectively, is not known. FCGBP is a cysteine-rich glycoprotein with an estimated M r of about 650k at least (the precursor comprises of 5405 amino acid residues; NCBI reference sequence: NP_003881.2; [46]) containing an N-terminal domain (FCGBP-N) followed by 12.5 repeats (R1-R12, and the shortened repeat R13s) arranged in tandem with similarity to D assembly domains (Figure 6A; for details, see [27]). The latter were originally detected in von vWF and later on in different gel-forming mucins from Xenopus laevis (frog integumentary mucin FIM-B.1) to human (MUC2, MUC5AC, MUC5B, MUC6) [33,35,47]. During multimerization of both vWF and the gel-forming mucins, an inter-molecular disulfide linkage is formed between D3 assemblies [35,47]. D3 dimerization of vWF requires Ca 2+ , and Ca 2+ -binding sites were identified in most of its vWD domains [47]. These coordination sites for Ca 2+ are conserved in vWD domains in both gel-forming mucins [34] and FCGBP ( Figure 6B). As a hallmark, D assemblies contain 4 cysteine-rich modules, i.e., von Willebrand D domain (vWD, eight cysteines), C8 (eight cysteines), TIL (trypsin inhibitor-like, 10 cysteines), and E (6 cysteines) [47]. These four modules pack in a highly circular fashion against one another in each D assembly and the vast majority of disulfide bridges are within a single cysteine-rich module [47].  [27]. The repeats R3-R5, R6-R8, and R9-R11 are parts of longer repeats (I, II, III). Additional cysteine residues (C) in R1 and R12 are shown in red (for details, see [27]). Scale bar: 1000 amino acid residues. (B) Depicted is a canonical repeat (R) consisting mainly of cysteine-rich vWD, C8, TIL, and E' modules (for details, see [27]). The number of cysteine residues is given above each module including additional cysteine residues N-terminal to vWD (3 C) and between vWD and C8 (2 C), respectively. In the vWD module, the six conserved cysteine residues are numbered and the disulfide bridges are indicated as deduced from vWF [47]. Also shown are the six conserved coordination sites for Ca 2+ within the vWD modules as deduced from vWF [47]. Outlined also are the conserved CXXC and CXXS motifs.
In FCGBP, each of the repeats R1-R12 contains assembly domains built up of somewhat modified versions of vWD (mostly six cysteines), C8, TIL, and E modules ( Figure 6); whereas R13s consists of a vWD domain only (for details, see [27]). By analogy with vWF [47], vWD domains in FCGBP are expected to be stabilized by three conserved disulfide bridges in the order C 1-5 , C 2-6 , and C 3-4 ( Figure 6B). Furthermore, all 13 vWD modules contain a conserved C 4 GL/AC 5 G motif [27,46], known as a CXXC motif (see Figure 6B), which is essential for the catalysis of redox reactions in thiol:disulfide oxidoreductases [48,49]. The conserved CGLCG motif is also present in porcine submaxillary mucin and MUC5AC, where it plays a role for N-terminal multimerization [43]. In addition, all TIL domains (10 cysteine residues) in FCGBP possess a conserved C 3 XXS motif ( Figure 6B; for details, see [27]), which is missing in vWF, but is a characteristic constituent of many disulfide isomerases, such as AGR2 [50], and the disulfide catalyst quiescin sulfhydryl oxidase 1 (QSOX1) [51]. Taken together, these are indications that FCGBP might be involved in disulfide isomerization reactions and covalent disulfide crosslinking.
As a hallmark, 11 of the 13 vWD domains (R1 to R11) contain the motif (W)GD↓PHY ( Figure 6A), which is subject to the autocatalytic cleavage between D and P ( Figure 6B), the preceding W residue accelerating the cleavage reaction [27]. Cleavage of this motif was first documented for rat Fcgbp in 2002 [52]. Remarkably, this motif is lacking in vWF. Similar cleavages of WGD↓PHY motifs occur in the vWD4 domains of the mucins MUC2 and MUC5AC [53,54] as well as in a variety of other proteins, such as toxin proteins from Gram-negative pathogens [55] and repulsive guidance molecules [56]. Cleavage can occur late in the secretory pathway, preferentially at a pH below 6 [53], and is probably Ca 2+ dependent [55]. This is the typical milieu of mucin storage granules, which is characterized by increased [H + ] and [Ca 2+ ] [57]. The pH dependence might explain why the processing of FCGBP is changed in prostate secretion of Atp12a-deficient mice, where the  [27]. The repeats R3-R5, R6-R8, and R9-R11 are parts of longer repeats (I, II, III). Additional cysteine residues (C) in R1 and R12 are shown in red (for details, see [27]). Scale bar: 1000 amino acid residues. (B) Depicted is a canonical repeat (R) consisting mainly of cysteine-rich vWD, C8, TIL, and E' modules (for details, see [27]). The number of cysteine residues is given above each module including additional cysteine residues N-terminal to vWD (3 C) and between vWD and C8 (2 C), respectively. In the vWD module, the six conserved cysteine residues are numbered and the disulfide bridges are indicated as deduced from vWF [47]. Also shown are the six conserved coordination sites for Ca 2+ within the vWD modules as deduced from vWF [47]. Outlined also are the conserved CXXC and CXXS motifs.
In FCGBP, each of the repeats R1-R12 contains assembly domains built up of somewhat modified versions of vWD (mostly six cysteines), C8, TIL, and E modules ( Figure 6); whereas R13s consists of a vWD domain only (for details, see [27]). By analogy with vWF [47], vWD domains in FCGBP are expected to be stabilized by three conserved disulfide bridges in the order C 1-5 , C 2-6 , and C 3-4 ( Figure 6B). Furthermore, all 13 vWD modules contain a conserved C 4 GL/AC 5 G motif [27,46], known as a CXXC motif (see Figure 6B), which is essential for the catalysis of redox reactions in thiol:disulfide oxidoreductases [48,49]. The conserved CGLCG motif is also present in porcine submaxillary mucin and MUC5AC, where it plays a role for N-terminal multimerization [43]. In addition, all TIL domains (10 cysteine residues) in FCGBP possess a conserved C 3 XXS motif ( Figure 6B; for details, see [27]), which is missing in vWF, but is a characteristic constituent of many disulfide isomerases, such as AGR2 [50], and the disulfide catalyst quiescin sulfhydryl oxidase 1 (QSOX1) [51]. Taken together, these are indications that FCGBP might be involved in disulfide isomerization reactions and covalent disulfide crosslinking.
As a hallmark, 11 of the 13 vWD domains (R1 to R11) contain the motif (W)GD↓PHY ( Figure 6A), which is subject to the autocatalytic cleavage between D and P ( Figure 6B), the preceding W residue accelerating the cleavage reaction [27]. Cleavage of this motif was first documented for rat Fcgbp in 2002 [52]. Remarkably, this motif is lacking in vWF. Similar cleavages of WGD↓PHY motifs occur in the vWD4 domains of the mucins MUC2 and MUC5AC [53,54] as well as in a variety of other proteins, such as toxin proteins from Gram-negative pathogens [55] and repulsive guidance molecules [56]. Cleavage can occur late in the secretory pathway, preferentially at a pH below 6 [53], and is probably Ca 2+ dependent [55]. This is the typical milieu of mucin storage granules, which is characterized by increased [H + ] and [Ca 2+ ] [57]. The pH dependence might explain why the processing of FCGBP is changed in prostate secretion of Atp12a-deficient mice, where the acidification of prostate fluid is disturbed [58]. Of note, after proteolytic cleavage, the FCGBP fragments are still cross-linked by disulfide bridges under non-reducing conditions [27,42,46,52], probably due to the disulfide bridge C 1-5 in the vWD modules ( Figure 6B). C 1 is in close proximity to the (W)GD↓PHY cleavage site and C 5 is part of the CXXC 5 motif ( Figure 6B). This might be a sign that the disulfide bridge C 1-5 becomes shear-sensitive particularly after proteolytic cleavage, when it changes from an intra-to an inter-chain bridge. Furthermore, the conserved six coordination sites for Ca 2+ are also located in close proximity to both the (W)GD↓PHY motif (one coordination site) and the CXXC motif (five coordination sites, Figure 6B). Taken together, this is a strong indication for a Ca 2+ dependent cleavage followed by a conformational change, which would allow the N-terminally generated proline residue to be deeply buried in the protein core as shown for a repulsive guidance molecule [56]. Furthermore, the β-carboxyl group of the C-terminal aspartate generated after the cleavage could be covalently linked to the ε-amino group of an internal lysine by nucleophilic attack [55]. Here, very stable isopeptide bonds could create crosslinks, either within the same molecule or neighboring FCGBP molecules. However, FCGBP does not crosslink to mucins, as claimed previously [42].
In vWF, the D1 and D2 assemblies are a prerequisite for intracellular packing of the precursor in Weibel-Palade bodies and the D'D3 assembly is required for multimerization of vWF via linkage of two free cysteines (C-1099, C-1142) [47]. These interactions require Ca 2+ and low pH [47]. In order to prevent premature inter-molecular disulfide formation, the two free cysteine residues are not surface exposed [47]. FCGBP contains 435 cysteine residues and this odd number suggests the presence of at least one free thiol, allowing the formation of an inter-molecular disulfide bridge [27]. Based on a comparison of the 13 repeats in FCGBP [27], three cysteine residues might be candidates for free cysteines ( Figure 6A), i.e., the additional C-593 in R1 (in vWD1), the additional C-4853 in R12 (just before vWD12), and one of the three N-terminal cysteines (C-449, C-455, C-464) in R1 (just before vWD1). Theoretically, R13 could also be involved in the formation of disulfide bridges [27]. The three proposed cysteine residues in R1 and R12 ( Figure 6A) could be particularly involved in the formation of inter-molecular disulfide bridges with TFF3 and with another FCGBP molecule, respectively. This would allow the formation of a variety of different FCGBP dimers and oligomers.

TFF3-FCGBP Forms Oligomers and Does Not Bind to DMBT1gp 340
The M r of TFF3-FCGBP can be roughly estimated by extrapolation after separation by non-denaturing AgGE and the use of commercial protein markers (see Section 4.3). Typically, the observed M r is far more than 300k [27] and a graphical extrapolation revealed values of about 6-8 × 10 6 . This is a clear indication that TFF3-FCGBP forms oligomers, which may consist of about 10 monomeric units. The observed formation of TFF3-FCGBP oligomers is in agreement with a recent report [59].
TFF3 with its single free C-terminal cysteine residue would be ideally suited to controlling FCGBP oligomerization by acting as a free thiol scavenger for FCGBP; this could limit the size of the FCGBP oligomers and also influence their structure (linear, circular, branched). In addition, the expected lectin activity of TFF3 could stabilize the TFF3-FCGBP oligomers; the latter would be an ideal extracellular matrix playing a key role in the innate immune defense of mucous epithelia, in addition to the network of gel-forming mucins. Such a mechanism would require the expression of both FCGBP and TFF3 in about equal molar amounts. This has been demonstrated in the past [27]. Protection by TFF3-FCGBP is of particular importance during inflammatory processes. In agreement with this, both TFF3 and FCGBP are upregulated by interleukin-13 (IL-13) [60][61][62].
DMBT1gp 340 is a glycoprotein typically expressed in many mucous epithelia including the submucosal glands of the bronchi, where it plays an important role in innate immune defense [63]. For example, it binds to lipopolysaccharides and various microorganisms (bacteria as well as viruses), and also interacts with surfactant proteins [63]. Of special note, DMBT1gp 340 was described as a TFF3 binding protein in vitro [64]. However, the results presented in Figure 5 argue against the association of TFF3-FCGBP with DMBT1gp 340 in bronchial extracts as well as BAL fluid. In addition, the mucin MUC5AC also does not seem to be associated with TFF3-FCGBP in the respiratory tract ( Figure 5). The latter would be in line with a recent report excluding the covalent linkage of FCGBP and the intestinal mucin MUC2 [42]. Taken together, TFF3-FCGBP seems to be an independent high-molecular-mass constituent of the respiratory mucus, probably with a role in innate immunity.

Respiratory Tract Secretory Specimens Contain 60k and 30k TFF3 Heterodimers
In bronchial secretions ( Figure 2D) as well as in BAL fluid ( Figure 4E), a TFF3 immunoreactive 60k-band appeared after SDS-PAGE under non-reducing conditions which released monomeric TFF3 forms after reduction ( Figure 3D). After purification of the 60kband from a bronchial secretion (Figure 3), TFF3 could be identified by proteome analysis directly in the 60k-band ( Figure 3E) as well as after its reductive release ( Figure 3E). Thus, it can be concluded that TFF3 is disulfide-linked to a partner protein with an M r of about 53k. Unfortunately, it was not possible to identify the partner protein unambiguously. It should be mentioned that a similar 60k-band was recently identified in the human stomach representing a disulfide-linked heterodimeric form of TFF1 [36], the latter having high similarity with TFF3 [4]. However, identification of the partner protein is also still missing [36]. Theoretically, one could propose that the 60k heterodimeric form of TFF3 might be a degradation product of TFF3-FCGBP. However, we could not find indications supporting this hypothesis. Nevertheless, and remarkably, in the course of characterizing the 60k-band, multiple tryptic fragments of the mucins MUC5AC and MUC5B were identified, all clustering in the N-terminal regions of these mucins (MUC5AC: within the 685 N-terminal amino acid residues of the precursor; MUC5B: within the 676 N-terminal amino acid residues of the precursor; data not shown). Currently, it cannot be excluded that in bronchial secretions, minute amounts of TFF3 might form disulfide-linked heterodimers with N-terminal fragments of MUC5AC and MUC5B. However, these proteomic results are an indication that in bronchial secretions both MUCAC and MUC5B are proteolytically cleaved at least at the N-terminal. The observed degradation is in line with the results from Figure 5, where respiratory MUC5AC has a much lower M r than gastric MUC5AC.
Furthermore, in bronchial secretions ( Figure 2E), as well as in BAL fluid ( Figure 4F) minute amounts of a TFF3 form with a M r of about 30k was detectable. This is expected to represent a heterodimeric TFF3 form with a disulfide-linked partner protein with an estimated M r of about 23k. Attempts to identify the partner protein failed.

Degradation of TFF3 in Bronchial Secretions
One of the hallmarks of the respiratory secretions (bronchial secretions and BAL fluid, respectively) is the occurrence of two TFF3 entities after reducing SDS-PAGE (Figures 2  and 4). Here, in addition to the normal form, a shortened TFF3 form appeared, the latter missing the N-terminal four amino acid residues EEYV, as shown for a bronchial secretion by proteome analysis ( Figure 3E). Also, BAL fluid contains an N-terminally shortened TFF3 entity ( Figure 4H). In contrast, in bronchial tissue extracts the shortened TFF3 form is missing.
A proteolytic degradation seems to be the most reasonable explanation for generating the shortened TFF3-form. However, this is rather unusual for TFF peptides, as the cysteine-rich TFF domains are well known for their resistance to proteolytic degradation [2]. Currently, the nature of this enzymatic process is not clarified. One potential candidate would be the neutrophil elastase, which is a serine protease released from neutrophils, the latter representing the typical inflammatory cells in the lung [65,66]. Generally, proteases play a major role in chronic lung diseases [67]. Neutrophil elastase has a relatively broad substrate specificity and preferentially cleaves after valine, cysteine, alanine, methionine and isoleucine residues (P1 position) [68]. Thus, the observed cleavage after Val-4 (Figure 3E) would be in agreement with the cleavage of TFF3 by the neutrophil elastase. Of note, in human bronchial secretion, TFF3 seems to be protease resistant in the TFF3-FCGBP heterodimer as well as the homo-dimeric form, whereas monomeric TFF3 predominantly appears in the truncated form ( Figure 2A). Thus, dimerization seems to protect TFF3 from proteolytic cleavage, perhaps by masking the potential cleavage site. The lack of proteolytic degradation in the tissue specimens (Figure 1) might be due to the lack of inflammation in these samples.
Currently, there are no convincing data defining unambiguously the biological role(s) of monomeric and homo-dimeric TFF3 or the shortened TFF3. Generally, TFF3 (and probably also fragments thereof) do not seem to support the restitution of mucous epithelia significantly as their motogenic and anti-apoptotic effects are rather weak [3], and could result from degradation of the TFF3-FCGBP heterodimer or from being a side product of incomplete heterodimer formation. Based on the various reports on the anti-apoptotic effects of TFF3 [3,69], one might speculate that TFF3 protects alveolar type 2 (AT2) cells from apoptosis. This could be due to a lectin-triggered receptor blocking/activation [4,62] by TFF3 and could have medical significance, as AT2 cells not only orchestrate pulmonary innate immunity and secrete a surfactant, but also act as progenitor cells for the alveolar epithelium [70]. Such a mechanism might be of clinical importance, particularly in the development of COPD [70]. However, TFF3 was also reported to increase apoptosis (proapoptotic effect [71]). Furthermore, monomeric TFF3 with its free thiol could act as a protective scavenger for ROS similar to that proposed for TFF1 [36,37,72]. By analogy with reports concerning the cervical mucus, TFF3 might even also affect the rheological properties of the respiratory mucus [19]. All patient-related procedures-bronchoscopies and surgical resections-were performed with a clinical indication, i.e., peripheral lung cancer (T1 to T2). All patients were smokers or former smokers and suffered from concomitant COPD GOLD Stages I to III. Patients were included when a visible mucous hypersecretion as a sign of chronic bronchitis was present. Patients with predominant emphysema without bronchial hypersecretion were excluded.

Human Specimens
Lung tissue was obtained from resected specimens from patients undergoing thoracic surgery because of peripheral lung cancer. Specimens were included in the study only, when they were free from malignancy, and not used for pathological workup.
BS and BAL were obtained during flexible bronchoscopy under local anesthesia. BS were collected by aspiration via the working channel of the bronchoscope into a special secretion trap. BAL was performed in the right middle lobe by rinsing and aspiring with five aliquots of 20 mL sterile saline.

SDS-PAGE, Agarose Gel Electrophoresis, and Western Blot Analysis
All methods were described in previous publications in detail, i.e., denaturing SDS-PAGE under reducing or non-reducing conditions, protein staining with Bio-Safe Coomassie Stain G-250 without fixation, non-denaturing AgGE, and periodic acid-Schiff (PAS) staining of mucins (dot blot) [27,75,77]. As a relative standard for non-denaturing AgGE, the GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) was used, as most commercial protein markers do not cover the Mr range above 300k, which is typical of TFF3-FCGBP (Figure 7). Staining of the DNA ladder was done with GelRed ® (41011, Biotium, Fremont, CA, USA). Western blot analysis after SDS-PAGE or AgGE was performed as reported [74,75,77,78]. Gels after non-reducing SDS-PAGE were subjected to post-in-gel reduction before blotting, as published previously [75].

Identification of Proteins by Bottom-Up Proteomics
For protein identification, gel bands were subjected to tryptic digestion, followed by liquid chromatography coupled to electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS), and resulting data were processed and analyzed with a search engine as described [76].

Tryptic In-Gel Digestion
Proteins in gel bands were digested according to Shevchenko et al. [80]. Briefly, 100% acetonitrile (ACN) and 100 mM NH 4 HCO 3 were used for shrinking and swelling, respectively. Disulfide bonds of the proteins in the gel-band were reduced with 10 mM dithiothreitol in a 100 mM NH 4 HCO 3 buffer. Alkylation of the SH-groups of cysteines was undertaken with 55 mM iodacetamide dissolved in 100 mM NH 4 HCO 3 . Tryptic digestion of the proteins in the gel bands were performed at 37 • C for 16 h in a NH 4 HCO 3 buffer (50 mM) containing 10% ACN and 8 ng/µL trypsin (sequencing-grade). The tryptic peptides were transferred into the supernatant with 2% formic acid (FA), 80% ACN. The solvent of the supernatant was removed by evaporation in a vacuum centrifuge. Prior to the LC-MS/MS analysis, the dried peptides were dissolved in 20 µL 0.1% FA (sample application buffer, solvent A).

Identification of Proteins by Bottom-Up Pro
For protein identification, gel bands we liquid chromatography coupled to electrospr (LC-ESI-MS/MS), and resulting data were p as described [76].

Tryptic In-Gel Digestion
Proteins in gel bands were digested acco acetonitrile (ACN) and 100 mM NH4HCO3 w tively. Disulfide bonds of the proteins in the threitol in a 100 mM NH4HCO3 buffer. Alky dertaken with 55 mM iodacetamide dissolve the proteins in the gel bands were performe (50 mM) containing 10% ACN and 8 ng/µL tides were transferred into the supernatant w vent of the supernatant was removed by eva LC-MS/MS analysis, the dried peptides wer cation buffer, solvent A).

Identification of Proteins by Bottom-Up Proteomics
For protein identification, gel bands were subjected to tryptic digestion, followed by liquid chromatography coupled to electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS), and resulting data were processed and analyzed with a search engine as described [76].

Tryptic In-Gel Digestion
Proteins in gel bands were digested according to Shevchenko et al. [80]. Briefly, 100% acetonitrile (ACN) and 100 mM NH4HCO3 were used for shrinking and swelling, respectively. Disulfide bonds of the proteins in the gel-band were reduced with 10 mM dithiothreitol in a 100 mM NH4HCO3 buffer. Alkylation of the SH-groups of cysteines was undertaken with 55 mM iodacetamide dissolved in 100 mM NH4HCO3.
Tryptic digestion of the proteins in the gel bands were performed at 37 °C for 16 hours in a NH4HCO3 buffer (50 mM) containing 10% ACN and 8 ng/µL trypsin (sequencing-grade). The tryptic peptides were transferred into the supernatant with 2% formic acid (FA), 80% ACN. The solvent of the supernatant was removed by evaporation in a vacuum centrifuge. Prior to the LC-MS/MS analysis, the dried peptides were dissolved in 20 µL 0.1% FA (sample application buffer, solvent A).

LC-MS/MS Data Processing and Protein Identification
LC-MS/MS data were processed with the software Proteome Discoverer 2.4.1.15 (Thermo Fisher Scientific, Bremen, Germany). Proteins were identified by using the search engine Sequest HT and the protein database SwissProt (www.uniprot.org, 2016). The following search parameters were used: Species: homo sapiens, precursor mass tolerance: 10 ppm; fragment mass tolerance: 0.2 Da. Missed cleavages: two were allowed; fixed modification: carbamidomethylation on cysteine residues; variable modification: oxidation of methionine residues. FDR: 1% using Percolator. For a reliable identification, at least two unique peptides per protein were used.
, Thermo Fisher Scientific, Bremen, Germany) and connected with an electrospray-ionization (ESI) source (fused-silica emitter: I.D. 10 µm; New Objective, Woburn, MA, USA; capillary voltage of 1650 V) to a trybrid mass spectrometer (MS) comprising a quadrupole, a linear trap and an orbitrap (Orbitrap Fusion, Thermo Fisher Scientific, Bremen, Germany). After washing the tryptic peptides for 5 min with 2% solvent B (0.1% FA in ACN) with 5 µL/min, they were separated using a flow rate of 200 nL/min with a gradient from 2% to 30% B in 30 min. The positive ion mode and data dependent acquisition mode (DDA) was used for mass spectrometry. Every second, over a m/z range from 400-1500 (resolution of 120,000 FWHM at m/z 200; transient length: 256 ms; maximum injection time: 50 ms; AGC target: 2 × 10 5 ), a MS scan was performed. For fragmentation, an HCD collision energy of 28%, an intensity threshold of 2 × 10 5 and an isolation width of 1.6 m/z was chosen. In the ion trap, MS/MS spectra were measured (scan-rate: 66 kDa/s; maximum injection time: 200 ms; AGC target: 1 × 10 4 ; underfill ratio of 10%; isolation width of 2 m/z).

LC-MS/MS Data Processing and Protein Identification
LC-MS/MS data were processed with the software Proteome Discoverer 2.4.1.15 (Thermo Fisher Scientific, Bremen, Germany). Proteins were identified by using the search engine Sequest HT and the protein database SwissProt (www.uniprot.org, 2016). The following search parameters were used: Species: homo sapiens, precursor mass tolerance: 10 ppm; fragment mass tolerance: 0.2 Da. Missed cleavages: two were allowed; fixed modification: carbamidomethylation on cysteine residues; variable modification: oxidation of methionine residues. FDR: 1% using Percolator. For a reliable identification, at least two unique peptides per protein were used.

Conclusions and Medical Perspectives
Taken together, the oligomers of TFF3-FCGBP are the predominant TFF3 form in the human respiratory tract. TFF3 might play a role in the oligomerization of FCGBP. TFF3-FCGBP is probably a key component of the disulfide-linked extracellular matrix with a major role in mucosal innate immune defense. For example, FCGBP could influence the adherence of microorganisms as well as their clearing [3]. FCGBP has even been proposed to act as a trap for viral-antibody complexes [81]. It could also influence the metastasis of tumors; however, the observations are contrary [40]. The application of TFF3, TFF3-FCGBP, FCGBP or FCGBP fragments, e.g. during BAL, or a drug-mediated modulation of FCGBP expression might be novel strategies to support the innate immune defense of the respiratory tract (but also of other mucous epithelia) against microbial infections including viruses, such as SARS-CoV-2.