Identification of four alternatively spliced transcripts of the Ucma/GRP gene, encoding a new Gla-containing protein.
The Ucma protein (Upper zone of growth plate and cartilage matrix associated protein) has recently been described as a novel secretory protein mainly expressed in cartilage and also as a novel vitamin-K-dependent protein named GRP (Gla-rich protein). This protein has the highest Gla content of any protein known to date. In this article, we identify four alternatively spliced variants of Ucma/GRP gene transcripts in mouse chondrocytes. We show that the expression of all four isoforms is associated with the early stages of chondrogenesis. The Ucma/GRP gene encodes four proteins named Ucma/GRP-F1, -F2, -F3, and -F4, which differ by exon 2, exon 4, or both. Among them, only Ucma/GRP-F1 and -F3 were secreted into the culture medium of transfected chondrocytes, while Ucma/GRP-F2 and -F4 accumulated in the cells. Using HeLa cells or freshly isolated embryonic mouse chondrocytes transfected with enhanced green fluorescent protein fusions, microscopy analysis revealed that Ucma/GRP-F1 and -F3 were localized in the Golgi complex, whereas Ucma/GRP-F2 and -F4 formed aggregates. This aggregation was microtubule-dependent since disruption of microtubules with nocodazole reduced Ucma/GRP-F2 and -F4 aggregation in a reversible manner. Using biochemical fractionation and Western blot analysis, Ucma/GRP-F1 and -F3 isoforms were detected in the soluble fraction while Ucma/GRP-F2 and -F4 were found in an insoluble-enriched fraction. We conclude that the co-expression of soluble and insoluble isoforms also Gla-rich and Gla-deleted isoforms may be finely tuned. Imbalance in isoform expression may therefore be involved in skeletal pathology.
Le Jeune
Marion
M
CNRS UMR 8161, Institut de Biologie de Lille, Université de Lille Nord de France, Institut Pasteur de Lille, 59021 Lille IFR 142, France.
Tomavo
Nathalie
N
Tian
Tian V
TV
Flourens
Anne
A
Marchand
Nathalie
N
Camuzeaux
Barbara
B
Mallein-Gerin
Frédéric
F
Duterque-Coquillaud
Martine
M
D015055:1-Carboxyglutamic Acid / Q000032:analysis*; D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology; D002454:Cell Differentiation / Q000502:physiology; D019902:Chondrocytes / Q000378:metabolism; D020219:Chondrogenesis / Q000502:physiology; D003593:Cytoplasm / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005091:Exons / Q000235:genetics; D016326:Extracellular Matrix Proteins; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008822:Mice, Transgenic; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D015388:Organelles / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D051789:Proto-Oncogene Protein c-fli-1 / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology
D055396:Bone Morphogenetic Protein 2; D016326:Extracellular Matrix Proteins; C497426:Fli1 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D020033:Protein Isoforms; D011506:Proteins; D051789:Proto-Oncogene Protein c-fli-1; D011993:Recombinant Fusion Proteins; D053773:Transforming Growth Factor beta1; C528958:Ucma protein, mouse; D015055:1-Carboxyglutamic Acid; D015739:Nocodazole