Biomedical subjects

M Domowicz

Publications and source records attributed to M Domowicz.

7 recordsLinked to original sources

PubMed1998

A member of a family of sulfate-activating enzymes causes murine brachymorphism.

Sulfation is critical to the function of a wide variety of biomolecules. This common modification requires the enzymatic synthesis of an activated sulfate donor, phosphoadenosine-phosphosulfate (PAPS). In higher organisms PAPS synthesis is catalyzed by a bifunctional sulfurylase kinase (SK) polypeptide having both ATP-sulfurylase and adenosine-phosphosulfate kinase activities. We report the identification of a gene family encoding murine SK proteins with these two activities. A family member, SK2, colocalizes with the locus for the autosomal recessive murine phenotype brachymorphism. Brachymorphic mice have normal lifespans, but abnormal hepatic detoxification, bleeding times, and postnatal growth, the latter being attributed to undersulfation of cartilage proteoglycan. A missense mutation in the SK2 coding sequence of bm mice that alters a highly conserved amino acid residue destroys adenosine-phosphosulfate kinase activity and therefore the ability of SK2 to synthesize PAPS. We conclude that a family of SK genes are responsible for sulfate activation in mammals, that a mutation in SK2 causes murine brachymorphism, and that members of this gene family have nonredundant, tissue-specific roles.

Amino Acid Sequence↗

PubMed1996

Age-dependent inhibition of neural crest migration by the notochord correlates with alterations in the S103L chondroitin sulfate proteoglycan.

In avian embryos, the notochord inhibits neural crest migration, resulting in the absence of neural crest cells from the perinotochordal space. Here, we test whether temporal changes in the ability of the notochord to inhibit neural crest migration correlate with alterations in the S1O3L chondroitin sulfate proteoglycan (CSPG). Because CSPGs are abundant in the perinotochordal space and the inhibitory effects of the notochord are chondroitinase sensitive both in vivo and in vitro, we examined the distribution and biochemical nature of a large CSPG whose core protein is recognized by the S103L antibody. The S103L CSPG is specific to the perinotochordal space during the course of neural crest migration and codistributes with the HNK-1 carbohydrate. Biochemical characterization reveals that the S103L CSPG bears the HNK-1 epitope and is the only HNK-l immunoreactive proteoglycan present around the notochord at these stages. Following neural crest migration, the S103L CSPG staining is maintained in the perinotochordal region and also is expressed later in cartilage. In 4-day-old embryos, however, the S103L CSPG undergoes a reduction of HNK-1 immunoreactivity. To examine the temporal nature of the notochord's inhibitory ability, we assayed the effects, on neural crest migration of grafting notochords from 2- to 5-day-old donor quail embryos into 2-day-old host chick embryos. Donor notochords from 2- to 3-day-old embryos inhibit neural crest cell migration, whereas the degree of inhibition is reduced or absent when notochords are derived from > or = 4-day-old donors. This suggests that older notochords lose their inhibitory ability. Interestingly, preincubation of younger notochords with the HNK-1 antibody blocks the inhibitory effect, suggesting that glycosylation of the perinotochordal matrix may be important. The time when the notochord loses its inhibitory ability as assessed by our in vivo grafting assay correlates with the biochemical and immunocytochemical changes in the notochordal S103L antigen. These data suggest that a species of S103L CSPG, which is expressed by the early notochord and bears the HNK-1 epitope, may be important for the inhibition of neural crest migration.

Animals↗

PubMed1996

S103L reactive chondroitin sulfate proteoglycan (aggrecan) mRNA expressed in developing chick brain and cartilage is encoded by a single gene.

A large chondroitin sulfate proteoglycan (CSPG) identified in embryonic chick brain, and synthesized exclusively by neurons in a developmentally expressed pattern that coincides with migration and establishment of neuronal nuclei, reacts with a monoclonal antibody (S103L) developed against the cartilage-specific CSPG, aggrecan. The relationship of the brain and cartilage S103L CSPGs was established by chemical, biosynthetic and molecular analyses. Significant posttranslational differences (absence of keratan sulfate (KS), less CS, and different sulfation patterns) distinguish the brain S103L species from the cartilage S103L species. However, quantitative and qualitative Northern analysis, cassette RT-PCR and direct cloning and sequencing of the entire brain-specific S103L CSPG coding sequence, all indicate that the brain and cartilage core proteins are identical. Thus, although the S103L CSPG synthesized by chick brain and cartilage are the product of a single gene, they are clearly biochemically distinct and differentially expressed proteoglycan products, suggesting tissue specific roles for these proteoglycan homologs.

Aggrecans↗

PubMed1996

The nanomelic mutation in the aggrecan gene is expressed in chick chondrocytes and neurons.

We have established the presence of at least two large chondroitin sulfate proteoglycans in the developing chick brain, one that reacts exclusively with HNK-1, a carbohydrate epitope found on several neural specific molecules, and one that reacts with S103L, a defined peptide epitope in the CS-2 domain of the cartilage-specific chondroitin sulfate proteoglycan (CSPG), aggrecan. In order to determine the relationships between the two distinct S103L-reactive CSPGs from cartilage (chondrocytes) and brain (neurons), as well as among the three large CSPGs expressed in brain, S103L, HNK-1 and versican, we studied the expression of these multiple proteoglycan species in the brain of nanomelic chicks. We have previously shown that homozygous embryos expressing the nanomelic phenotype exhibit a single point mutation in the aggrecan gene. In the present study, the S103L CSPG is not accumulated or synthesized by embryonic chick CNS tissue or E8CH neuronal cultures derived from nanomelic chick embryo cerebral hemispheres. In contrast, expression of both versican and the HNK-1 CSPG was normal in the mutant embryo CNS. Pulse chase experiments demonstrated the presence of the 380 kDa precursor in normal neurons and the 300 kDa truncated precursor in nanomelic neurons. Northern blot analysis revealed normal-sized mRNA but reduced levels of expression of the S103L CSPG message in nanomelic neurons, while expression of the versican message was comparable in normal and nanomelic neurons. Most conclusively, the point mutation previously identified in nanomelic cartilage mRNA was also identified in nanomelic brain mRNA. Together these results provide evidence that a single aggrecan gene is expressed in both cartilage and CNS tissue leading to the production of identical core proteins which then undergo differential and tissue-specific post-translation processing, resulting in the characteristic tissue-specific proteoglycans. Furthermore, versican and the HNK-1 CSPG, although structurally and chemically similar to the S103L CSPG, are the products of separate genes.

Animals↗

PubMed1996

Brain aggrecan.

During development, the extracellular matrix (ECM) is a complex dynamic structure whose components and organization help to establish the requisite position and state of differentiation. Until recently, the large chondroitin sulfate proteoglycan, aggrecan, has been localized predominantly to skeletal tissue and considered a hallmark of cartilage differentiation. We have identified the presence of aggrecan in two other highly differentiated systems, brain and notochord, with clearly distinct expression patterns. In chick cartilage, aggrecan starts to be expressed at embryonic day 5 in limb rudiments, continues through the entire period of chondrocyte development, and remains a biochemical marker of the cartilage phenotype thereafter. In brain, aggrecan has a very low level of expression beginning at day 7, increases up to day 13, markedly decreases after day 16, and is not expressed posthatching. This pattern coincides with migration and establishment of neuronal nuclei in the chick telencephalon and has been proposed to be a component of the migration arrest mechanism. In very primitive embryos, aggrecan is detected as early as stage 16 in the notochord, long before chondrogenesis occurs, is then expressed up to day 5 and decreases thereafter. The expression of aggrecan occurs during the time of active neural crest migration and through the onset of sclerotomal differentiation, and correlates with the notochords' ability to inhibit neural crest cell migration. Animal models defective in aggrecan biosynthesis have been invaluable in delineating these functions. In addition we have characterized these proteoglycans by chemical, biosynthetic, and molecular analyses. Although significant post-translation differences distinguish the cell-specific aggrecan species, their core proteins are the products of a single gene. Our findings of the expression of the same gene (aggrecan) in multiple ontogenously unrelated differentiating tissue systems and at different times over the developmental life of an organism provide an elegant model system to study the regulation and interplay in expression of that gene, as well as the effect of alterations in that single gene simultaneously in several developing programs.

Aggrecans↗

PubMed1995

The isolation and characterization of cDNA encoding the mouse bifunctional ATP sulfurylase-adenosine 5'-phosphosulfate kinase.

Biosynthesis of the activated sulfate donor, adenosine 3'-phosphate 5'-phosphosulfate, involves the sequential action of two enzyme activities: ATP sulfurylase, which catalyzes the formation of adenosine 5'-phosphosulfate (APS) from ATP and free sulfate, and APS kinase, which subsequently phosphorylates APS to produce adenosine 3'-phosphate 5'-phosphosulfate. Oligonucleotide primers were derived from a human infant brain-expressed sequence tag putatively encoding a portion of APS kinase. Using these primers, reverse transcriptase-polymerase chain reaction was performed on mRNA from neonatal normal mice resulting in amplification of a 127-bp DNA fragment. This fragment was subsequently used to screen a mouse brain lambda gt11 cDNA library, yielding a 2.2-kb clone. Primers were designed from the 5'-end of the 2.2-kb clone, and 5'-rapid amplification of cDNA ends was used to obtain the translation start site. Sequence from the overlapping clones was assembled into a 2475-bp composite sequence, which contains a single open reading frame that translates into a 624-deduced amino acid sequence. Northern blots of total RNA from neonatal mice yielded a single message species at approximately 3.3 kb. Southern blot of genomic DNA digested with several restriction enzymes suggested the gene is present as a single copy. Comparison against sequence data bases suggested the composite sequence was a fused sulfurylase-kinase product, since the deduced amino acid sequence showed extensive homology to known separate sequences of both ATP sulfurylase and APS kinase from several sources. The first 199 amino acids corresponded to APS kinase sequence, followed by 37 distinct amino acids, which did not match any known sequence, followed by 388 amino acids that are highly homologous to known ATP sulfurylase sequences. Finally, recombinant enzyme expressed in COS-1 cells exhibited both ATP sulfurylase and APS kinase activity.

Amino Acid Sequence↗

PubMed1995

The biochemically and immunologically distinct CSPG of notochord is a product of the aggrecan gene.

Using the monoclonal antibody S103L, which reacts specifically with an epitope in the chondroitin sulfate-rich domain of the chick cartilage chondroitin sulfate proteoglycan (CSPG) core protein, we have identified the predominant CSPG expressed by notochord. This large notochord CSPG is first detected immunohistochemically as early as stage 16, long before chondrogenesis occurs, and is expressed continuously during the time of active neural crest migration and through the onset of sclerotomal differentiation. Because of the cross-reactivity of both notochord and cartilage CSPGs with the S103L antibody, extensive molecular and biochemical analysis of the two CSPGs was carried out. Striking differences distinguish the notochord and cartilage (aggrecan) CSPGs at the level of posttranslational modification. Notably, cartilage aggrecan carries a significant content of keratan sulfate (KS) chains, while the notochord CSPG is devoid of KS. In contrast, cartilage aggrecan lacks the HNK-1 epitope, while the notochord CSPG has a high content of HNK-1. Three different approaches were used to establish the relationship of the two CSPGs at the molecular level. Northern blot analysis, using aggrecan probes, detected same-sized messages from notochord and cartilage RNA. Overlapping fragments, generated by RT-PCR using primers covering 98% of the entire coding sequence from the known cartilage structure, were of identical size in notochord and cartilage. Taking advantage of our recent studies, which demonstrated a single base change in the aggrecan gene resulting in conversion of Glu to a STOP codon in exon 12 of chick aggrecan as the molecular basis of the defect nanomelia, we demonstrated that the same mutation was present in notochord mRNA from nanomelic chicks. These results provide evidence that the chick aggrecan gene is expressed very early in development in notochord and confirm that the core proteins expressed in chick notochord and cartilage are derived from the same gene. These findings strongly support the hypothesis that the final structural characteristics of each proteoglycan are determined not only by the core protein but also by tissue-specific, developmentally regulated posttranslational mechanisms, functioning within the context of the requirement for specific extracellular matrices.

Aggrecans↗