Temperature-sensitive Glycosaminoglycan Biosynthesis in a Chinese Hamster Ovary Cell Mutant Containing a Point Mutation in Glucuronyltransferase I

Ge, Wei; Bai, Xiaomei

doi:10.1074/jbc.m311621200

Cited by 5 publications

(3 citation statements)

References 37 publications

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“…As previously shown, artificially introduced missense mutations can reduce enzymatic activity and secondarily decreased stability of the mutant, dysfunctional enzyme by ER retention and subsequent proteasomal decay. 25 To test whether the mutation p.Arg277Gln indeed reduces GlcAT-I activity by impaired enzymatic function and not by primary reduced protein level, we transfected cells with the human GlcAT-I wild-type and the mutated (p.Arg277Gln) vector. We found a marked reduction of GlcAT-I activity in the mutant despite similar amounts of the wild-type and mutant proteins ( Figures 3C-3E), suggesting that the reduced enzymatic activity of GlcAT-I affects protein stability.…”

Section: Reduced Glcat-i Enzyme Activity In Patient Cellsmentioning

confidence: 99%

Faulty Initiation of Proteoglycan Synthesis Causes Cardiac and Joint Defects

Baasanjav

Al‐Gazali

Hashiguchi

et al. 2011

The American Journal of Human Genetics

107

119

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Proteoglycans are a major component of extracellular matrix and contribute to normal embryonic and postnatal development by ensuring tissue stability and signaling functions. We studied five patients with recessive joint dislocations and congenital heart defects, including bicuspid aortic valve (BAV) and aortic root dilatation. We identified linkage to chromosome 11 and detected a mutation (c.830G>A, p.Arg277Gln) in B3GAT3, the gene coding for glucuronosyltransferase-I (GlcAT-I). The enzyme catalyzes an initial step in the synthesis of glycosaminoglycan side chains of proteoglycans. Patients' cells as well as recombinant mutant protein showed reduced glucuronyltransferase activity. Patient fibroblasts demonstrated decreased levels of dermatan sulfate, chondroitin sulfate, and heparan sulfate proteoglycans, indicating that the defect in linker synthesis affected all three lines of O-glycanated proteoglycans. Further studies demonstrated that GlcAT-I resides in the cis and cis-medial Golgi apparatus and is expressed in the affected tissues, i.e., heart, aorta, and bone. The study shows that reduced GlcAT-I activity impairs skeletal as well as heart development and results in variable combinations of heart malformations, including mitral valve prolapse, ventricular septal defect, and bicuspid aortic valve. The described family constitutes a syndrome characterized by heart defects and joint dislocations resulting from altered initiation of proteoglycan synthesis (Larsen-like syndrome, B3GAT3 type).

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Section: Reduced Glcat-i Enzyme Activity In Patient Cellsmentioning

confidence: 99%

Faulty Initiation of Proteoglycan Synthesis Causes Cardiac and Joint Defects

Baasanjav

Al‐Gazali

Hashiguchi

et al. 2011

The American Journal of Human Genetics

107

119

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“…Chinese hamster ovary (CHO) cell mutants deficient in xylosyltransferase 2 ( Xylt2 ), galactosyltransferase I ( β4galt7 ), and glucuronosyltransferase I ( β 3 gat3 ) were generated previously by chemical mutagenesis (3–5). The pgsA745 cell line harbors a nonsense mutation in Xylt2 (6), completely lacks HS and CS/DS, and has been used by many laboratories to assess the role of GAGs in various processes, including adhesion and invasion by pathogens (7).…”

Section: Introductionmentioning

confidence: 99%

Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion

Wijk

Döhrmann

Hallström

et al. 2017

mBio

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To understand the role of glycosaminoglycans in bacterial cellular invasion, xylosyltransferase-deficient mutants of Chinese hamster ovary (CHO) cells were created using clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-cas9) gene targeting. When these mutants were compared to the pgsA745 cell line, a CHO xylosyltransferase mutant generated previously using chemical mutagenesis, an unexpected result was obtained. Bacterial invasion of pgsA745 cells by group B Streptococcus (GBS), group A Streptococcus, and Staphylococcus aureus was markedly reduced compared to the invasion of wild-type cells, but newly generated CRISPR-cas9 mutants were only resistant to GBS. Invasion of pgsA745 cells was not restored by transfection with xylosyltransferase, suggesting that an additional mutation conferring panresistance to multiple bacteria was present in pgsA745 cells. Whole-genome sequencing and transcriptome sequencing (RNA-Seq) uncovered a deletion in the gene encoding the laminin subunit α2 (Lama2) that eliminated much of domain L4a. Silencing of the long Lama2 isoform in wild-type cells strongly reduced bacterial invasion, whereas transfection with human LAMA2 cDNA significantly enhanced invasion in pgsA745 cells. The addition of exogenous laminin-α2β1γ1/laminin-α2β2γ1 strongly increased bacterial invasion in CHO cells, as well as in human alveolar basal epithelial and human brain microvascular endothelial cells. Thus, the L4a domain in laminin α2 is important for cellular invasion by a number of bacterial pathogens.

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“…This rodent MT model should be largely free of mechanical, dietary, vascular and systemic hormonal influences, save those impacted directly or indirectly by tissue temperature. Incubation temperature is already known to affect osteoblast activity (21), glycosaminoglycan synthesis (22), and collagen calcification (23) in vitro; however, these studies were conducted at the cellular level, whereas our experiments, that rely on a Mouse tail length (A) and body mass (B) measured at weekly intervals beginning at weaning. Means Ϯ 1 SEM shown (n ϭ 7/group).…”

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Temperature regulates limb length in homeotherms by directly modulating cartilage growth

Serrat

King

Lovejoy

2008

Proc. Natl. Acad. Sci. U.S.A.

130

121

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Allen's Rule documents a century-old biological observation that strong positive correlations exist among latitude, ambient temperature, and limb length in mammals. Although genetic selection for thermoregulatory adaptation is frequently presumed to be the primary basis of this phenomenon, important but frequently overlooked research has shown that appendage outgrowth is also markedly influenced by environmental temperature. Alteration of limb blood flow via vasoconstriction/vasodilation is the current default hypothesis for this growth plasticity, but here we show that tissue perfusion does not fully account for differences in extremity elongation in mice. We show that peripheral tissue temperature closely reflects housing temperature in vivo, and we demonstrate that chondrocyte proliferation and extracellular matrix volume strongly correlate with tissue temperature in metatarsals cultured without vasculature in vitro. Taken together, these data suggest that vasomotor changes likely modulate extremity growth indirectly, via their effects on appendage temperature, rather than vascular nutrient delivery. When combined with classic evolutionary theory, especially genetic assimilation, these results provide a potentially comprehensive explanation of Allen's Rule, and may substantially impact our understanding of phenotypic variation in living and extinct mammals, including humans.Allen's Rule ͉ bone growth ͉ bone tissue culture ͉ cartilage biology ͉ thermoregulation E cogeographical rules relating climate to extremity length and body mass have long been central tenets of biology, and are among the best supported observations in natural-dwelling species (1). Allen's Rule codifies the observation that appendages (ears, limbs, and tails) of animals living in cold geographical regions are consistently shorter than those of closely related counterparts occupying warmer climes (2). Shortened extremities minimize heat loss by reducing surface area relative to volume and have long been viewed as genetically determined thermoregulatory adaptations (1). However, the heritability of extremity length is largely unknown, because similar phenotypes can be reproduced in laboratory mammals by modifying their ambient rearing temperature (3-6) (Fig. 1). The mechanism by which environmental temperature modulates extremity growth has remained elusive (7,8). Understanding growth plasticity is critical to basic evolutionary analyses, because many characters that have long been hypothesized to be adaptations may instead be partial or even entirely effects of ambient temperature (9, 10). Moreover, knowledge of this phenotypic plasticity will be a key factor in ecological conservation strategies for anticipated changes in global climate that may have direct impacts on human economics and sustainability (11).The traditional explanation for temperature-growth effects in skeletal extremities is an altered supply of essential nutrients and growth factors via increased or decreased blood flow that results from changes in vasomotor tone (i.e., t...

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Temperature-sensitive Glycosaminoglycan Biosynthesis in a Chinese Hamster Ovary Cell Mutant Containing a Point Mutation in Glucuronyltransferase I

Cited by 5 publications

References 37 publications

Faulty Initiation of Proteoglycan Synthesis Causes Cardiac and Joint Defects

Faulty Initiation of Proteoglycan Synthesis Causes Cardiac and Joint Defects

Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion

Temperature regulates limb length in homeotherms by directly modulating cartilage growth

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