Biomedical subjects

Nancy B Schwartz

Publications and source records attributed to Nancy B Schwartz.

6 recordsLinked to original sources

PubMed2005

APBP-1, a DNA/RNA-binding protein, interacts with the chick aggrecan regulatory region.

Expression of the extracellular proteoglycan aggrecan is both cell-specific and developmentally regulated. Previous studies identified six functionally defined cis elements in the aggrecan promoter region which were shown to repress aggrecan gene expression (1). Using competition electrophoretic mobility shift assays (EMSAs) we have now identified in nuclear extracts a functional repressor cis element, (T/C)TCCCCT(A/C)RRC, which occurs at multiple locations within the chick aggrecan regulatory region. We purified the factor that binds to this cis element and established that it, APBP-1 (aggrecan promoter-binding protein-1), is a 19-kDa protein that has significant homology to CIRP (cold inducible RNA-binding protein). Recombinantly expressed APBP-1 mimics the native cis element-trans factor interaction in EMSAs. In situ hybridization demonstrates that aggrecan and APBP-1 RNA expression are restricted to complementary tissues in the developing limb, and Northern blot analysis of chick limb bud mRNA shows that APBP-1 mRNA expression is inversely correlated with aggrecan mRNA expression. Functional analyses by transient transfections and Northern blot analyses suggest APBP-1 has the capacity to repress aggrecan expression, indicating that this factor may be important regulator of aggrecan gene expression.

Aggrecans↗

PubMed2004

Proteoglycans in brain development.

Proteoglycans, as part of the extracellular or cell-surface milieu of most tissues and organ systems, play important roles in morphogenesis by modulating cell-matrix or cell-cell interactions, cell adhesiveness, or by binding and presenting growth and differentiation factors. Chondroitin sulfate proteoglycans which constitute the major population of proteoglycans in the central nervous system may influence formation of neuronal nuclei, establishment of boundaries for axonal growth and act as modulators of neuronal outgrowth during brain development, as well as during regeneration after injury. There is a paucity of information on the role of chondroitin sulfate proteoglycans in central nervous system organogenesis. In the chick embryo, aggrecan has a regionally specific and developmentally regulated expression profile during brain development. By Northern and Western blot analysis, aggrecan expression is first detected in chick brain on embryonic day 7 (E7), increases from E7 to E13, declines markedly after E16, and is not evident in hatchling brains. The time course and pattern of aggrecan expression observed in ventricular zone cells suggested that it might play a role in gliogenesis. We have analyzed the role of aggrecan during brain development using a aggrecan-deficient model, nanomelia. In nanomelic chicks, expression and levels of neurocan and brevican is not affected, indicating a non-redundant role for these members of the aggrecan gene family. Our analysis of the aggrecan-deficient model found a severely altered phenotype which affects cell behavior in a neuronal culture paradigm and expression of astrocytic markers in vivo . Taken together our results suggest a function for aggrecan in the specification of a sub-set of glia precursors that might give rise to astrocytes in vivo.

Animals↗

PubMed2003

Aggrecan regulates telencephalic neuronal aggregation in culture.

Proteoglycans have been suggested to play roles in pattern formation in the developing central nervous system. In the chick embryo, aggrecan, a chondroitin sulfate proteoglycan, has a regionally-specific and developmentally-regulated expression profile. Telencephalic neuronal cultures, when aggregated, exhibit aggrecan expression patterns comparable to those observed in vivo. The chicken mutation nanomelia produces a truncated aggrecan species that cannot be processed further and is not secreted. Neurons from normal and nanomelic chick embryo telencephalon were scored for aggregate formation and analyzed for distribution of aggrecan protein and expression of aggrecan mRNA. Distinctly different pattern formation, with respect to aggregate size (smaller) and number (fewer) were observed in poly-L-lysine plated neuronal cultures derived from nanomelic embryos when compared to those derived from normal embryos. Significantly, the nanomelic phenotype was subsequently rescued upon addition of the brain-specific form of aggrecan. Modulation of neuronal aggregate formation was mimicked by treatment with chondroitinase ABC but not other glycanases, and was rescued by addition of chondroitin 6-sulfate to the culture media. Lastly, although broad and diffuse distribution of aggrecan among the cell aggregates in the culture paradigm was observed by immunocytochemistry, mRNA in situ hybridization revealed that only a small population of cells in the center of the aggregates was responsible for the production of the secreted aggrecan found associated with neuronal aggregates. These studies suggest a function for aggrecan as a diffusible signal in CNS histomorphogenesis.

Aggrecans↗

PubMed2003

Developmental expression of the HNK-1 carbohydrate epitope on aggrecan during chondrogenesis.

Previously, we showed that the HNK-1 carbohydrate epitope is expressed on aggrecan synthesized in the notochord but not in mature cartilage. In the present study, we demonstrate that in immature cartilage (embryonic day 6) the HNK-1 epitope is also expressed predominantly on aggrecan proteoglycan molecules. This finding was verified by using an aggrecan-deficient mutant, the nanomelic chick, which lacks HNK-1 immunostaining in the extracellular matrix of dividing and hypertrophic chondrocytes as late as embryonic day 12. By using both biochemical and immunologic approaches, the initially prominent expression of the HNK-1 epitope is down-regulated as development of limb and vertebral cartilage proceeds, so that by embryonic day 14 no HNK-1 is detectable. Localization changes with development and the HNK-1-aggrecan matrix becomes restricted to dividing and hypertrophic chondrocytes and is particularly concentrated in the intraterritorial matrix. Concomitant with the temporal and spatial decreases in HNK-1, there is a significant increase in keratan-sulfate content and the aggrecan-borne HNK-1 epitope is closely associated with proteolytic peptides that contain keratan sulfate chains, rather than chondroitin sulfate chains or carbohydrate-free domains. Lastly, the diminution in HNK-1 expression is consistent with a reduction in mRNA transcripts specific for at least one of the key enzymes in HNK-1 oligosaccharide biosynthesis, the HNK-1 sulfotransferase. These findings indicate that the HNK-1 carbohydrate may be a common modifier of several proteoglycans (such as aggrecan) that are usually expressed early in development, and that HNK-1 addition to these molecules may be regulated by tissue- and temporal-specific expression of requisite sulfotransferases and glycosyltransferases.

Aggrecans↗

PubMed2002

Identification and functional characterization of the novel BM-motif in the murine phosphoadenosine phosphosulfate (PAPS) synthetase.

PAPS synthetase (SK) catalyzes the two sequential reactions of phosphoadenosine phosphosulfate (PAPS) synthesis. A functional motif in the kinase domain of mouse SK, designated the BM-motif ((86) LDGDNhRxhh(N/S)(K/R)(97)), was defined in the course of identifying the brachymorphic (bm) defect. Sequence comparison and the secondary structure predicted for APS kinase suggest that the BM-motif consists of a DGD-turn sequence flanked by other conserved residues. Mutational analysis of the DGD-turn revealed that a flexible and neutral amino acid is preferred at residue 88, that negatively charged residues are strictly required at positions 87 and 89, and that the active site is rigid. The reduction in kinase activity for all DGD-turn mutants, except G88A, was much less severe than the reduction in overall activity, indicating that the BM-motif may also be playing a role in adenosine phosphosulfate (APS) channeling. Two switch mutations, LD86DL and DN89ND, designed to test the positional constraints of Asp(87) and Asp(89), exhibited complete loss of both kinase and overall activities, while LD86DL also exhibited a significant (60%) loss of reverse sulfurylase activity, suggesting that this peptide region is interacting with the sulfurylase domain as well as functioning in the kinase reaction. Other residues targeted for mutational analysis were the highly conserved flanking Asn(90), Arg(92), and Lys(97). N90A resulted in a partial (30%) loss in kinase and overall activities, R92A exhibited total loss of kinase and overall activities, and K97A had no effect on any of the three activities. The complexity of the bifunctional SK in catalyzing the kinase reaction and channeling APS is illustrated by the strict requirements of this novel structural motif in the kinase active site.

Amino Acid Motifs↗

PubMed2002

Chondrodysplasias due to proteoglycan defects.

The proteoglycans, especially the large chondroitin sulfate proteoglycan aggrecan, have long been viewed as important components of the extracellular matrix of cartilage. The drastic change in expression during differentiation from mesenchyme to cartilage, the loss of tissue integrity associated with proteoglycan degradation in several disease processes and, most important, the demonstration of abnormalities in proteoglycan production concomitant with the aberrant growth patterns exhibited by the brachymorphic mouse, the cartilage matrix deficient mouse, and the nanomelic chick provide the strongest evidence that the proteoglycan aggrecan is essential during differentiation and for maintenance of the skeletal elements. More recently, mutations associated with proteoglycans other than aggrecan, especially the heparan sulfate proteoglycans, glypican and perlecan, suggest an important role for these molecules in skeletal development as well. This review focuses on the molecular bases of the hereditary proteoglycan defects in animal models, as well as of some human chondrodysplasias, that collectively are providing a better understanding of the role of proteoglycans in the development and maintenance of the skeletal elements.

Animals↗