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.
Domowicz
Miriam S
MS
Department of Pediatrics, The University of Chicago, Chicago, Illinois, USA. mdxx@midway.uchicago.edu
Mueller
Melissa M
MM
Novak
Todd E
TE
Schwartz
Lauren E
LE
Schwartz
Nancy B
NB
D053674:Aggrecans; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000196:embryology; D002356:Cartilage / Q000196:embryology / Q000378:metabolism; D002642:Chick Embryo; D019902:Chondrocytes / Q000378:metabolism; D002810:Chondroitinases and Chondroitin Lyases / Q000494:pharmacology; D003001:Cloning, Molecular; D015536:Down-Regulation; D010450:Endopeptidases / Q000378:metabolism; D000939:Epitopes; D016326:Extracellular Matrix Proteins; D005121:Extremities / Q000196:embryology; D018507:Gene Expression Regulation, Developmental; D014453:Glucuronosyltransferase / Q000378:metabolism; D006026:Glycoside Hydrolases; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015238:Sulfotransferases / Q000096:biosynthesis*; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D001616:beta-Galactosidase / Q000494:pharmacology
D053674:Aggrecans; D000939:Epitopes; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D009844:Oligosaccharides; D011509:Proteoglycans; D012333:RNA, Messenger; D014453:Glucuronosyltransferase; C109318:HNK-1 sulfotransferase; D015238:Sulfotransferases; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase; D010450:Endopeptidases; D014357:Trypsin; D002810:Chondroitinases and Chondroitin Lyases