The chondrodystrophy, nanomelia: biosynthesis and processing of the defective aggrecan precursor

Abstract

The lethal chicken mutation nanomelia leads to severe skeletal defects because of a deficiency of aggrecan, which is the largest aggregating chondroitin sulphate proteoglycan of cartilage. In previous work, we have demonstrated that nanomelic chondrocytes produce a truncated aggrecan precursor that fails to be secreted, and is apparently arrested in the endoplasmic reticulum (ER). In this study, we investigated the biosynthesis and extent of processing of the abnormal aggrecan precursor. The truncated precursor was translated directly in cell-free reactions, indicating that it does not arise post-translationally. Further studies addressed the processing capabilities of the defective precursor. We found that the mutant precursor was modified by N-linked, mannose-rich oligosaccharides and by the addition of xylose, but was not further processed; this is consistent with the conclusion that it moves no further along the secretory pathway than the ER. Using brefeldin A we demonstrated that the defective precursor can function as a substrate for Golgi-mediated glycosaminoglycan chains, but does not do so in the nanomelic chondrocyte because it fails to be translocated to the appropriate membrane compartment. These studies illustrate how combined cell biological/biochemical and molecular investigations may contribute to our understanding of the biological consequences and molecular basis of genetic diseases, particularly those involving errors in large, highly modified molecules such as proteoglycans.

Regulation of chondrocyte differentiation by the actin cytoskeleton and adhesive interactions

“…Aggrecan and other proteoglycans provide a cushioning role of the matrix, but also act to immobilize and store growth factors and thereby function as molecular organizers of the ECM and cartilage in general (Rouslahti and Yamaguchi, 1991). The importance of these pathways for skeletal development is shown by several human diseases caused by mutations in the genes encoding Sox9, Collagen II, and Aggrecan, or regulators of GAG sulfation (Eyre et al, 1986;Spranger et al, 1994;Vertel et al, 1994;Wagner et al, 1994;Chan et al, 1995;Hastbacka et al, 1996;Bi et al, 2001;Rossi and Superti-Furga, 2001;Gleghorn et al, 2005).…”

Role of the C-terminal G3 Domain in Sorting and Secretion of Aggrecan Core Protein and Ubiquitin-mediated Degradation of Accumulated Mutant Precursors

“…The nanomelic chick bears a mutation in the aggrecan gene that introduces an early stop codon into the translated sequence, resulting in synthesis of a truncated core protein, which is neither glycosylated nor secreted by chondrocytes (1). The mutant precursor is modified by addition of N-linked oligosaccharides and the chondroitin sulfate chain initiating xylose, but does not acquire mature CS chains, consistent with the conclusion that it progresses no further than the endoplasmic reticulum (ER) in the secretory pathway (2). These studies suggested a previously unrecognized role for the C-terminal globular domain: that it might contain recognition or retention signals or that proper folding of this specific domain, which may involve interactions with molecular chaperones, is necessary to effect exit of the entire core protein precursor from the ER.…”

“…From our studies, which elucidated the mutation in the nanomelic chick, it was learned that not only was there a complete lack of aggrecan in the mutant extracellular matrix, there was also an accumulation of a truncated, partially processed precursor in the chondrocyte ER, leading to the hypoth- esis that the deleted G3 domain was involved in the intracellular maturation and secretion process of this proteoglycan (1,2). In the present study, we focused mainly on: (i) identifying the minimal alteration from the native structure tolerated before aggrecan processing and secretion are affected, (ii) elucidating the pathway and players involved in the early steps of processing, (iii) investigating whether the unnatural accumulation of unprocessed core protein induces a stress-related response, and (iv) determining the mode of degradation of the ER-accumulated product.…”

Adaptation of Sensory Neurons to Hyalectin and Decorin Proteoglycans

“…2 H). Previous work has shown that chondrocytes derived from chickens with the nanomelic mutation synthesize a truncated form of the aggrecan protein that is not processed or secreted (Vertel et al, 1994;Domowicz et al, 1995Domowicz et al, , 1996Domowicz et al, , 2000Chen et al, 2002) and that S103L-aggrecan immunoreactivity is not observed in the notochord of nanomelic animals (Domowicz et al, 2003). Aggrecan is known to be differentially processed in different tissues (Domowicz et al, 1995), raising the possibility that neural tissue in nanomelic mutants may secrete a truncated form of aggrecan that is uniquely modified compared with chondrocytes.…”