Mucin 21/epiglycanin modulates cell adhesion

Abstract

The molecular structure of mouse Mucin 21 (Muc21)/epiglycanin is proposed to have 98 tandem repeats of 15 amino acids and three exceptional repeats with 12 or 13 amino acids each, followed by a stem domain, a transmembrane domain, and a cytoplasmic tail. A cDNA of Muc21 having 84 tandem repeats of 15 amino acids was constructed and transfected using a Venus vector into HEK 293T cells. The fluorescent cells, which were considered to express Muc21, were nonadherent. This antiadhesion effect was lessened when constructs with smaller numbers of tandem repeats were used, suggesting that the tandem repeat domain plays a crucial role. Cells expressing Muc21 were significantly less adherent to each other and to extracellular matrix components than control cells. Antibody binding to the cell surface integrin subunits alpha5, alpha6, and beta1 was reduced in Muc21 transfectants in a tandem repeat-dependent manner, whereas equal amounts of proteins were detected by Western blot analysis. Muc21 was expressed as a large glycoprotein that was highly glycosylated with O-glycans at the cell surface, as detected by flow cytometry, Western blotting, and lectin blotting. Although at least a portion of Muc21 was glycosylated with sialylated glycans, removal of sialic acid did not influence the prevention of adhesion.

FIGURE 1.

Muc21/epiglycanin is assumed to have 98 TR of 15 amino acids. Muc21 consists of three exons. Exon I encodes a signal sequence; exon II encodes the end of signal sequence, a TR domain, a neck domain, an SEA-like domain, and a transmembrane (TM) domain; and exon III encodes a cytoplasmic (CYP) domain. Exon I was discovered by 5′-RACE, and the full size of the TR domain was estimated by the results of 5′-RACE, NCBI database analysis, and partial sequencing of Muc21 gene from a BAC clone.

FIGURE 2.

Artificial Muc21/epiglycanin cDNA having 84 TR was constructed and expressed in 293T cells. A, methods of constructing the Muc21 expression vector are summarized. B, cells transiently transfected with the Muc21-IRES2-Venus vector were stained with mAb 1A4-1 and analyzed by flow cytometry using allophycocyanin-conjugated streptavidin as a fluorescence group at an excitation wavelength 638 nm and emission wavelength 660/20 nm.

FIGURE 3.

Expression of the Muc21 resulted in large highly glycosylated glycoproteins with o-glycans. A, a mock (indicated as M) vector and a Muc21 expression vector having 84 TR and N-terminal FLAG tag (indicated as T) were transfected into 293T cells, and the cell lysates were precipitated with anti-FLAG antibody. The immunoprecipitates were separated on 4% polyacrylamide gels in the presence of 0.1% SDS, blotted onto polyvinylidene difluoride membranes, and detected with the same antibody or lectins. B, 293T cells transiently transfected with the Muc21-84TR-IRES2-Venus vector were stained with biotinylated PNA, VVA-B4, or SSA and analyzed by flow cytometry using biotinylated allophycocyanin-conjugated streptavidin as a fluorescence group at an excitation wavelength 638 nm and emission wavelength 660/20 nm.

FIGURE 4.

Muc21 expression in 293T cells induced morphologic changes. A, pictures of cells transiently transfected with mock or Muc21-84TR-IRES2-Venus vector are shown. Scale bar indicates 100 μm. B, the number of cells from the culture medium after transient transfection of Muc21-84TR-IRES2-Venus was evaluated against cells expressing the vectors as Venus-positive cells. C, the floating cells after transient expression of the N-terminal FLAG-tagged Muc21 vector were collected from culture medium and were subjected to TUNEL assays. The solid line, dashed line, and dotted line indicate the floating cells, negative control, and positive control, respectively.

FIGURE 5.

Long TR domain seemed to have a major effect on the antiadhesive property of Muc21. A, mutants lacking a cytoplasmic domain were constructed with an N-terminal FLAG tag and transfected into 293T cells. The number of floating cells in FLAG-positive cells was evaluated. B, extracellular domain mutants having various portions of Muc21 were constructed with an N-terminal FLAG tag and C-terminal c-myc tag and transfected into 293T cells. Note that the mutants in B were constructed using a different vector system from the mutants in A.

FIGURE 6.

Muc21 expression prevented homotypic cell-cell and cell-ECM interactions in a TR-dependent manner. A, mock-transfected cells (diamonds), cells transiently expressing Muc21 with 4 TR (triangles) or Muc21 with 84 TR (circles) were compared for homotypic aggregation. The cells were untreated (open symbols) or treated with sialidase (filled symbols) and sorted to collect Venus-positive populations. More than 90% Venus-positive cells were used. B, adhesion assays were performed with the same preparations of cells as in A. The six consecutive columns represent, from left to right, mock transfectants, sialidase-treated mock transfectants, cells transiently expressing Muc21 with 4 TR before and after sialidase treatment, and cells transiently expressing Muc21 with 84 TR before and after sialidase treatment. BSA, bovine serum albumin.

FIGURE 7.

Cell surface accessibilities of integrins α5, α6, and β1 were reduced by the expression of Muc21 with 84 TR. A, cells transiently transfected with mock, Muc21-4TR-IRES2-Venus, or Muc21-84TR-IRES2-Venus vectors were stained with anti-integrin α3, α5, α6, αV, or β1 antibodies, biotinylated second antibodies, and allophycocyanin-conjugated streptavidin and analyzed by flow cytometry. B, the adherent cells after transfection with mock or the Muc21-4TR-IRES2-Venus vector or the floating cells after transfection with the Muc21-84TR-IRES2-Venus vector were collected and lysed in lysis buffer. Cell lysates were separated electrophoretically on 7.5% polyacrylamide gels in the presence of 0.1% SDS and blotted onto polyvinylidene difluoride membranes. The blotted membranes were stained with anti-integrin α3, α5, α6, αV, or β1 antibodies or anti-β-tubulin antibodies. Although integrin subunits α3 and αV were below the control levels shown by the dots by flow cytometry, immunoprecipitates by the corresponding antibodies were observed by the Western blotting (WB).