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The epiphysis of developing bones is a cartilaginous structure that is eventually replaced by bone during skeletal maturation. We have separated a dermatan sulfate proteoglycan, epiphycan, from decorin and biglycan by using dissociative extraction of bovine fetal epiphyseal cartilage, followed by sequential ion-exchange, gel permeation, hydrophobic, and Zn 2؉ chelate chromatographic steps. Epiphycan is a member of the small leucine-rich proteoglycan family, contains seven leucine-rich repeats (LRRs), is related to osteoglycin to 34 kDa were more heterogeneous than those in other dermatan sulfate small leucine-rich proteoglycans and were found in the acidic N-terminal region of the protein core, N-terminal to the LRRs. A four-cysteine cluster was present at the N terminus of the LRRs, and a disulfide-bonded cysteine pair was present at the C terminus of the protein core. The seventh LRR and an N-linked oligosaccharide were between the two C-terminal cysteines. An additional potential N-glycosylation site near the C terminus did not appear to be substituted at a significant level.Cartilage contains a variety of proteoglycans. Particularly abundant are aggrecan and the small leucine-rich proteoglycans (SLRPs 1 ) (1) fibromodulin and decorin. Several other proteoglycans in cartilage have also been identified, including versican, perlecan, and the leucine-rich proteoglycans lumican and biglycan. The relative abundance of these proteoglycans varies during development and by location within the tissue. It is likely that this variation has a role in the differentiation and maintenance of tissue structure. The exact roles of the SLRPs are unclear at present, but it is thought that fibromodulin and decorin are involved in the process of collagen fibrillogenesis (2, 3) and may play a crucial role in optimizing the diameter of collagen fibrils that will eventually be replaced during remodeling of the cartilage during calcification. It is possible that the SLRPs also have a role in regulating growth factors, e.g. transforming growth factor-␤, which binds to decorin (4, 5).A hallmark of the SLRPs are the cysteine clusters that flank the leucine-rich repeats. These cysteines form disulfide bonds and perhaps provide a structure that differs from leucine-rich repeat (LRR)-containing proteins that do not contain this feature. The LRR motif was first identified by Patthy (6) and is characterized by an LXXLXLXXNXL sequence, where X is any amino acid and L is often a leucine, but may be any amino acid with a hydrophobic aliphatic side chain (Ile, Val, and Met). The LRR motif is conserved throughout evolution, and the increasing number of members of this family includes a range of proteins with diverse functions and distributions (reviewed by Kobe (7)). The three-dimensional structure of the porcine ribonuclease inhibitor, a member of the leucine-rich protein family, has been determined (8). In this protein, the 15 individual LRRs adopt a stacked ␤-sheet/␣-helix hairpin structure, resulting in an overall horseshoe shape and indicatin...

Objective-A missense mutation in the Microtubule Associated Serine/Threonine Like kinase gene (MASTL, FLJ14813) on human chromosome 10 was previously linked to a novel form of autosomal dominant inherited thrombocytopenia in a single pedigree. The mutation results in an amino acid change from glutamic acid at position 167 to aspartic acid and segregates perfectly with thrombocytopenic individuals within this extended family. The phenotype is characterized by mild thrombocytopenia with an average platelet count of 60,000 platelets per microliter of blood. We wanted to determine the expression and localization of MASTL, as well as its role in developing thrombocytes using an in vivo model system.Methods-Northern blot analysis allowed us to examine expression patterns. Morpholino knockdown assays in zebrafish (Danio rerio) were employed to determine in vivo contribution to thrombocyte development. Transient expression in BHK cells resulted in localization of both the wild type and E167D mutant forms of MASTL kinase to the nucleus.Results-Northern blot analysis indicates that MASTL mRNA is restricted in its expression to hematopoietic and cancer cell lines. A transient knockdown of MASTL in zebrafish results in deficiency of circulating thrombocytes. Transient expression of recombinant MASTL kinase in vitro demonstrates localization to the nucleus. Conclusions: Functional studies presented here demonstrate a direct relationship between the transient knockdown of the MASTL kinase expression and the reduction of circulating thrombocytes in zebrafish. This transient knockdown of MASTL in zebrafish correlates with a decrease in the expression of the thrombopoietin receptor, c-mpl, and the CD41 platelet adhesion protein, GpIIb, but has no effect on essential housekeeping zebrafish gene, EF1α.

1567 Microtubule-associated serine/threonine like kinase (MASTL kinase) was originally identified by genetic linkage as the probable mutant gene responsible for the platelet phenotype in a family with autosomal-dominant thrombocytopenia. MASTL kinase is the mammalian ortholog of Drosophila Greatwall kinase, which is required for cell cycle regulation. Affected members of the thrombocytopenic family carried a mutant MASTL kinase allele with a single nucleotide change that converted a glutamic acid codon to one specifying aspartic acid at amino acid 167 of the protein; no unaffected members of the family carried the mutant allele. Affected family members had a 60% reduction in the numbers of circulating platelets and megakaryocytes that were less mature and less polyploid than normal megakaryocytes. We have sought to determine how MASTL kinase affects platelet production and circulation by generating a strain of mice deficient in MASTL kinase. For this, we obtained embryonic stem (ES) cells containing a gene trap mutation within the murine Mastl gene from the Mutant Mouse Regional Resource Center of the National Center for Research Resources (MMRRC, University of California, San Diego). The mutation was generated by an insertional disruption, and the resulting mutant allele carries a beta–galactosidase/neomycin fusion expression cassette within the fourth intron of the Mastl kinase gene. Chimeric animals were bred and their offspring were genotyped by Southern blotting, confirming germ-line transmission. F1 heterozygote offspring were produced and appear developmentally normal and healthy, with platelet counts indistinguishable from those of their wild-type littermates. We have yet to observe a single weaned animal that is homozygous for the mutant allele in the F1 mice, indicating that MASTL kinase deficiency results in embryonic lethality. We observed decidual swellings at 13.5 days post coitus (dpc), which contained no traces of either embryos or yolk sac membranes. While this has made it impossible to genotype these malformed embryos, it does indicate that the embryos are viable to 3.5–5.5 dpc and are able to implant within the uterine horn. This unexpected phenotype demonstrates a significant and previously unknown role for Mastl kinase in embryonic development. We hypothesize that Mastl kinase may be involved in regulating cell differentiation during embryogenesis. In terms of role of MASTL kinase in familial thrombocytopenia, the mutant animals have allowed us to conclude that the thrombocytopenic syndrome is very unlikely to be the result of haploinsufficiency of MASTL kinase because heterozygous animals have normal platelet counts. Because of the dominant nature of the syndrome, this suggests that the mechanism involves either gain-of-function of the mutant allele or a dominant-negative inhibition of MASTL function specific to the megakaryocyte, two possibilities we are examining. Disclosures: No relevant conflicts of interest to declare.

Previously in our lab, a missense mutation in the MASTL (FLJ14813) gene on human chromosome 10 was linked to an autosomal dominantly inherited thrombocytopenia in a single pedigree. The mutation results in an amino acid change from glutamic acid at position 167 to aspartic acid and correlates with the inheritance of the disorder in all the affected individuals. The patients present with mild thrombocytopenia with an average platelet count of 60,000 platelets per microliter of blood. Northern blot analysis indicates that MASTL mRNA is restricted in its expression to hematopoietic and cancer cell lines. Functional studies presented here demonstrate a direct relationship between the transient knockdown of the Microtubule Associated Serine/Threonine Like (MASTL) kinase expression and the reduction of circulating thrombocytes in zebrafish (Danio rerio) while erythrocytes development normally. This transient knockdown of MASTL in zebrafish correlates with a decrease in the expression of the thrombopoietin receptor, c-mpl, and the CD41 platelet adhesion GpIIb protein but has no effect on essential housekeeping zebrafish gene, EF1α. The transient expression of a fluorescent protein fused to MASTL and E167D MASTL kinase demonstrates that MASTL localizes to the nucleus of cells as determined by confocal microscopy and TO-PRO-3 counterstain.