Mice deficient in biosynthetic enzymes for chondroitin sulfate

Because of space limitations, please refer to topics “Chondroitin sulfate/dermatan sulfate glycosaminoglycans: their biosynthetic machineries” and “Biosynthesis of the common tetrasaccharide sequence in the glycosaminoglycan-protein linker region” in this series, and comprehensive reviews (1, 2) for details on the gene names, aliases, and catalytic properties of individual biosynthetic enzymes for chondroitin sulfate (CS). Also, please note that the cited references (7-26) for each knockout (KO) mouse are described in Table 1.

The first direct evidence of physiological importance of CS chains was provided by the identification of nematode orthologue of chondroitin synthase (ChSy) as a causative gene for squashed valva (sqv) mutants that are characterized by perturbation of vulval invagination and by a cytokinetic failure in fertilized eggs (3, 4) (see “Glycosaminoglycans present in the model organism Caenorhabditis elegans”). In mice, gene KO of GlcAT-I, which leads to complete loss of two classes of sulfated glycosaminoglycans, CS and heparan sulfate (HS), is also embryonic lethal before the 8-cell stage due to cytokinetic failure (7). This phenotype is principally attributed to the loss of CS chains, because selective removal of CS chains in wild-type 2-cell embryos with a bacterial CS-degrading enzyme chondroitinase ABC also results in a lethal cell division defect (7). These observations indicate an indispensable role of CS chains in early embryonic cytokinesis.

In contrast to the GlcAT-I KO mice, all four single KO mice of enzymes involved in CS backbone synthesis, ChSy-1, ChPF, ChGn-1, and ChGn-2 are not embryonic lethal, but are viable and fertile, although they do show a reduced CS production and/or an imbalanced sulfation in the CS chains (8, 11-13, 15, 16). These discrepancies are probably due to the functional redundancy among these enzymes. However, ChSy-1 KO and ChGn-1 KO mice display subtle skeletal abnormalities including chondrodysplasia, decreased bone density, digit-patterning defects, and craniofacial abnormality (8, 12, 13, 15). Such skeletal phenotypes are feasible in terms of a remarkable abundance of CS-proteoglycans in cartilaginous tissues. Indeed, a cartilage-specific double KO of ChGn-1 and ChGn-2 in mice leads to a more severe skeletal deformation with a drastic CS reduction, and their systemic double KO mice exhibit postnatal lethality (16). Recently, a genome-edited KO of ChSy-3 in mice has also been reported to cause a spontaneous intervertebral disc degeneration (10). Furthermore, ChSy-1 has been identified as a causative gene in a mouse model with a spontaneous mutation that shows accelerated aging and age-related disease phenotypes including chronic inflammation and neurodegeneration (9). Interestingly, an apparently detrimental effect of CS chains on axonal regeneration after central nervous system injury (14) and their unexpected cardioprotective role at acute phase of heart failure (17) have been demonstrated using ChGn-1 KO and ChGn-2 KO mice, respectively.

The CS sugar backbone can be sulfated at various positions of the disaccharide units under strict control of multiple sulfotransferases including C4ST-1, C6ST-1, and GalNAc4S-6ST (see, topic “Chondroitin sulfate/dermatan sulfate glycosaminoglycans: their biosynthetic machineries” in this series). A gene trap mutation of C4ST-1 in mice causes severe chondrodysplasia, which leads to neonatal lethality mainly due to respiratory distress (18). By contrast, in the original study of mice deficient in C6ST-1, no apparent phenotypic abnormalities are reported, except for a marked reduction in the number of splenic naive T lymphocytes (19). However, a recent study revealed that C6ST-1 KO leads to keratinocyte hyperproliferation and impaired skin permeability (20). Notably, C6ST-1 transgenic mice, which have a higher abundance of 6-O-sulfated CS, retain ocular dominance neuronal plasticity even in adulthood (5), and exhibit a higher bone mineral density (6). Mice deficient in GalNAc4S-6ST are completely defective in CS/DS chains containing 4,6-O-disulfated disaccharide E units, are viable and show no apparent developmental abnormalities except for the decline in protease activities in bone marrow-derived mast cells (21). However, detained analyses revealed that they have abnormally low bone mass, largely due to impaired osteoblast differentiation (22). These findings strongly indicate the physiological significance of the specific sulfation in CS chains.

Notably, mice deficient in D4ST-1 or DS-epi1, both of which are specific enzymes for conversion of CS into its stereoisomer, DS, also display reduced body size, lower survival frequency, reduced fertility, and increased skin fragility (23, 24). These phenotypes are reminiscent of the those seen in functional deficiencies in the respective human counterparts that have been identified as causative genes for Ehlers-Danlos syndrome, musculocontractural types 1 and 2. Although mouse development is not obviously affected by a DS-epi2 single KO (25), double KO of DS-epi1 and DS-epi2 in mice display embryological defects and a high frequency of neonatal death (26). (see “Dermatan sulfate deficient mouse”).

Tadahisa Mikami & Hiroshi Kitagawa
(Laboratory of Biochemistry, Kobe Pharmaceutical University)

Jun. 15, 2023