Genomic analysis of a murine cell-surface sialomucin, MGC-24/CD164. (1/25)

MGC-24 is a sialomucin originally found in human gastric carcinoma cells, and in human hematopoietic progenitor cells. In the human, soluble and transmembrane forms of MGC-24 are present, and the transmembrane form has been implicated in adhesion of hematopoietic progenitor cells to marrow stroma cells. In the mouse, we found that only the transmembrane form was expressed in many organs. Northern blotting and in situ hybridization analysis showed that MGC-24 mRNA was widely expressed in various adult and embryonic tissues. The mouse MGC-24 gene, which we isolated, spanned about 12 kb and was comprised of six exons. The transmembrane domain and the cytoplasmic domain were encoded by a single exon; the finding agrees with the absence of an alternatively spliced product of mouse MGC-24. The minimal promoter of mouse MGC-24 was embedded in GC-rich sequences, in which two Sp1 binding motifs were found, but it lacked TATA and CAAT boxes. That the promoter resembles that of house-keeping genes is consistent with the broad expression of mouse MGC-24 mRNA.  (+info)

Endolyn is a mucin-like type I membrane protein targeted to lysosomes by its cytoplasmic tail. (2/25)

Endolyn (endolyn-78) is a membrane protein found in lysosomal and endosomal compartments of mammalian cells. Unlike 'classical' lysosomal membrane proteins, such as lysosome-associated membrane protein (lamp)-1, it is also present in a subapical compartment in polarized WIF-B hepatocytes. The structural features that determine sorting of endolyn are unknown. We have identified a rat endolyn cDNA by expression screening. The cDNA encodes a ubiquitously expressed type I membrane protein with a short cytoplasmic tail of 13 amino acids and many putative sites for N- and O-linked glycosylation in the predicted luminal domain. Endolyn is closely related to two human mucin-like proteins, multi-glycosylated core protein (MGC)-24 and CD164 (MGC-24v), expressed in gastric carcinoma cells and bone marrow stromal and haematopoietic precursor cells respectively. The predicted transmembrane and cytoplasmic tail domains of endolyn, as well as parts of its luminal domain, also show some similarities with lamp-1 and lamp-2. Like these and other known lysosomal membrane proteins, endolyn contains a YXXO motif at the C-terminus of its cytoplasmic tail (where O is a bulky hydrophobic amino acid), but with no preceding glycine. Nonetheless, the last ten amino acids of this tail, when transplanted on to human CD8, caused efficient targeting of the chimaeric protein to endosomes and lysosomes in transfected normal rat kidney cells.  (+info)

Functionally defined CD164 epitopes are expressed on CD34(+) cells throughout ontogeny but display distinct distribution patterns in adult hematopoietic and nonhematopoietic tissues. (3/25)

Three distinct classes of epitopes on human CD164 have been identified. Two of these, recognized by the monoclonal antibodies 105A5 and 103B2/9E10, are the CD164 class I and class II functionally defined epitopes, which cooperate to regulate adhesion and proliferation of CD34(+) cell subsets. In this article, we demonstrate that these 2 CD164 epitopes are expressed on CD34(+) cells throughout ontogeny, in particular on CD34(+ )cell clusters associated with the ventral floor of the dorsal aorta in the developing embryo and on CD34(+) hematopoietic precursor cells in fetal liver, cord blood, and adult bone marrow. While higher levels of expression of these CD164 epitopes occur on the more primitive AC133(hi)CD34(hi)CD38(lo/-) cell population, they also occur on most cord blood Lin(-)CD34(lo/-)CD38(lo/- )cells, which are potential precursors for the AC133(hi)CD34(hi)CD38(lo/-) subset. In direct contrast to these common patterns of expression on hematopoietic precursor cells, notable differences in expression of the CD164 epitopes were observed in postnatal lymphoid and nonhematopoietic tissues, with the class I and class II CD164 epitopes generally exhibiting differential and often reciprocal cellular distribution patterns. This is particularly striking in the colon, where infiltrating lymphoid cells are CD164 class I-positive but class II-negative, while epithelia are weakly CD164 class II-positive. Similarly, in certain lymphoid tissues, high endothelial venules and basal and subcapsular epithelia are CD164 class II-positive, while lymphoid cells are CD164 class I-positive. It therefore seems highly likely that these CD164 class I and II epitopes will mediate reciprocal homing functions in these tissue types.  (+info)

The ratio of splicing variants of MGC-24/CD164, a sialomucin, correlates with the metastatic potential of colorectal carcinomas. (4/25)

MGC-24/CD164 is a sialomucin expressed in many normal and cancerous tissues. In humans, soluble and transmembrane forms of MGC-24 are produced by alternative splicing. The total MGC-24 RNA level was found to be lower in human colorectal carcinomas as compared with the adjacent normal mucosal tissues. Lower MGC-24 mRNA levels in colon carcinomas and in the adjacent normal mucosa epithelium correlate with lymphatic vessel invasion by the carcinoma. The ratio of the soluble form to the transmembrane form of the mRNA in colorectal carcinomas was determined by ribonuclease protection assay. Higher ratios were correlated with less venous invasion and less remote metastasis, which became evident during postoperative observation.  (+info)

CD164 monoclonal antibodies that block hemopoietic progenitor cell adhesion and proliferation interact with the first mucin domain of the CD164 receptor. (5/25)

The novel sialomucin, CD164, functions as both an adhesion receptor on human CD34+ cell subsets in bone marrow and as a potent negative regulator of CD34+ hemopoietic progenitor cell proliferation. These diverse effects are mediated by at least two functional epitopes defined by the mAbs, 103B2/9E10 and 105A5. We report here the precise epitope mapping of these mAbs together with that of two other CD164 mAbs, N6B6 and 67D2. Using newly defined CD164 splice variants and a set of soluble recombinant chimeric proteins encoded by exons 1-6 of the CD164 gene, we demonstrate that the 105A5 and 103B2/9E10 functional epitopes map to distinct glycosylated regions within the first mucin domain of CD164. The N6B6 and 67D2 mAbs, in contrast, recognize closely associated and complex epitopes that rely on the conformational integrity of the CD164 molecule and encompass the cysteine-rich regions encoded by exons 2 and 3. On the basis of their sensitivities to reducing agents and to sialidase, O-sialoglycoprotease, and N-glycanase treatments, we have characterized CD164 epitopes and grouped them into three classes by analogy with CD34 epitope classification. The class I 105A5 epitope is sialidase, O-glycosidase, and O-sialoglycoprotease sensitive; the class II 103B2/9E10 epitope is N-glycanase, O-glycosidase, and O-sialoglycoprotease sensitive; and the class III N6B6 and 67D2 epitopes are not removed by such enzyme treatments. Collectively, this study indicates that the previously observed differential expression of CD164 epitopes in adult tissues is linked with cell type specific post-translational modifications and suggests a role for epitope-associated carbohydrate structures in CD164 function.  (+info)

Relationship between novel isoforms, functionally important domains, and subcellular distribution of CD164/endolyn. (6/25)

Functional analyses have indicated that the human CD164 sialomucin may play a key role in hematopoiesis by facilitating the adhesion of human CD34(+) cells to the stroma and by negatively regulating CD34(+)CD38(lo/-) cell proliferation. We have identified three novel human CD164 variants derived by alternative splicing of bona fide exons from a single genomic transcription unit. The predominant CD164(E1-6) isoform, encoded by six exons, is a type I transmembrane protein containing two extracellular mucin domains (I and II) interrupted by a cysteine-rich non-mucin domain. The 103B2/9E10 and 105A5 epitopes, which specify ligand binding characteristics, are located on the exon 1-encoded mucin domain I. Three human CD164(E1-6) mRNA species, exhibiting differential polyadenylation site usage, are differentially expressed in hematopoietic and non-hematopoietic tissues. This study provides additional evidence that human CD164(E1-6) represents the ortholog of murine MGC-24v and rat endolyn. Comparative analysis of murine MGC-24v/CD164(E1-6) with human CD164(E1-6) revealed two potential splice variants and a similar genomic structure. Whereas the human CD164 gene is located on chromosome 6q21, the mouse gene occurs in a syntenic region on chromosome 10B1-B2. By confocal microscopy, human CD164 in CD34(+)CD38(+) hematopoietic progenitor (KG1B) and epithelial cell lines appears to be localized primarily in endosomes and lysosomes, with low concentrations at the cell surface. However, in a minority of KG1B cells, CD164 is more prominently expressed at the plasma membrane and in the recycling endosomes, suggesting that its distribution is regulated in cells of hematopoietic origin.  (+info)

Identification of a role for the sialomucin CD164 in myogenic differentiation by signal sequence trapping in yeast. (7/25)

Determination and differentiation of skeletal muscle precursors requires cell-cell contact, but the full range of cell surface proteins that mediate this requirement and the mechanisms by which they work are not known. To identify participants in cell contact-mediated regulation of myogenesis, genes that encode secreted proteins specifically upregulated during differentiation of C2C12 myoblasts were identified by the yeast signal sequence trap method (K. A. Jacobs, L. A. Collins-Racie, M. Colbert, M. Duckett, M. Golden-Fleet, K. Kelleher, R. Kriz, E. R. La Vallie, D. Merberg, V. Spaulding, J. Stover, M. J. Williamson, and J. M. McCoy, Gene 198:289-296, 1997), followed by RNA expression analysis. We report here the identification of CD164 as a gene expressed in proliferating C2C12 cells that is upregulated during differentiation. CD164 encodes a widely expressed cell surface sialomucin that has been implicated in regulation of cell proliferation and adhesion during hematopoiesis. Stable overexpression of CD164 in C2C12 and F3 myoblasts enhanced their differentiation, as assessed by both morphological and biochemical criteria. Furthermore, expression of antisense CD164 or soluble extracellular regions of CD164 inhibited myogenic differentiation. Treatment of C2C12 cells with sialidase or O-sialoglycoprotease, two enzymes previously reported to destroy functional epitopes on CD164, also inhibited differentiation. These data indicate that (i) CD164 may play a rate-limiting role in differentiation of cultured myoblasts, (ii) sialomucins represent a class of potential effectors of cell contact-mediated regulation of myogenesis, and (iii) carbohydrate-based cell recognition may play a role in mediating this phenomenon.  (+info)

Competing sorting signals guide endolyn along a novel route to lysosomes in MDCK cells. (8/25)

We have examined the trafficking of the mucin-like protein endolyn in transfected, polarized MDCK cells using biochemical approaches and immunofluorescence microscopy. Although endolyn contains a lysosomal targeting motif of the type YXXPhi and was localized primarily to lysosomes at steady state, significant amounts of newly synthesized endolyn were delivered to the apical cell surface. Antibodies to endolyn, but not lamp-2, were preferentially internalized from the apical plasma membrane and efficiently transported to lysosomes. Analysis of endolyn-CD8 chimeras showed that the lumenal domain of endolyn contains apical targeting information that predominates over basolateral information in its cytoplasmic tail. Interestingly, surface polarity of endolyn was independent of O-glycosylation processing, but was reversed by disruption of N-glycosylation using tunicamycin. At all times, endolyn was soluble in cold Triton X-100, suggesting that apical sorting was independent of sphingolipid rafts. Our data indicate that a strong, N-glycan-dependent apical targeting signal in the lumenal domain directs endolyn into a novel biosynthetic pathway to lysosomes, which occurs via the apical surface of polarized epithelial cells.  (+info)

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