Tissue-specific changes in the hydroxylysine content and cross-links of collagens and alterations in fibril morphology in lysyl hydroxylase 1 knock-out mice.

We have generated mice with targeted inactivation of the Plod1 gene for lysyl hydroxylase 1 (LH1). Its human mutations cause Ehlers-Danlos syndrome VIA (EDS VIA) characterized by muscular hypotonia, joint laxity, and kyphoscoliosis. The Plod1(-/-) mice are flaccid and have gait abnormalities. About 15% of them died because of aortic rupture and smooth muscle cells in non-ruptured Plod1(-/-) aortas showed degenerative changes. Collagen fibrils in the Plod1(-/-) aorta and skin had an abnormal morphology. The LH activity level in the Plod1(-/-) skin and aorta samples was 35-45% of that in the wild type. The hydroxylysine content was decreased in all the Plod1(-/-) tissues, ranging from 22% of that in the wild type in the skin to 75 and 86% in the femur and lung. The hydroxylysylpyridinoline crosslinks likewise showed decreases in all the Plod1(-/-) tissues, ranging from 28 and 33% of that in the wild type in the aorta and cornea to 47 and 59% in femur and tendon, while lysylpyridinolines were increased. The hydroxylysines found in the Plod1(-/-) collagens and their cross-links were evidently synthesized by the other two LH isoenzymes. Few data are available on abnormalities in EDS VIA tissues other than the skin. Plod1(-/-) mice offer an in vivo model for systematic analysis of the tissue-specific consequences of the lack of LH1 activity and may also provide a tool for analyzing the roles of connective tissue in muscle function and the complex interactions occurring in the proper assembly of the extracellular matrix.

Molecular defects in the Ehlers-Danlos syndrome.

Several abnormalities in collagen biosynthesis have been described in patients with Ehlers-Danlos syndrome. Examples of collagen structural mutations as well as post-translational enzymatic defects have been detected. Patients with hydroxylysine-deficient collagen disease (Ehlers-Danlos type VI) have diminished lysyl hydroxylase activity. One mutant enzyme has been characterized which is thermally labile and had an altered affinity for ascorbate. Another mutant enzyme had a normal requirement for cofactors but activity was diminished. Type VII Ehlers-Danlos syndrome is associated with altered processing of procollagen to collagen. Most often the disorder is associated with deficient procollagen aminoprotease activity. One patient appears to represent a structural mutation of pro alpha 2 (I) resulting in incomplete cleavage of the amino terminal propeptide. One family with x-linked Ehlers-Danlos syndrome (type V) has been described with altered lysyl oxidase activity. Other patients with this disorder have had normal lysyl oxidase activity. The ecchymotic form of Ehlers-Danlos syndrome (type IV) has defective type III collagen-synthesis. Patients have been described with absent synthesis, diminished synthesis and diminished secretion.

Abnormal collagen in ataxia telangiectasia.

Ataxia telangiectasia has been associated with varying clinical and laboratory abnormalities of the cutaneous, immune, neurological, and endocrine systems. Recently, a primary defect in tissue differentiation has been proposed as a probable cause of the disease. The presence of biochemically abnormal collagen in the skin of two siblings with the disease may further support this hypothesis.

Abnormal properties of collagen lysyl hydroxylase from skin fibroblasts of siblings with hydroxylysine-deficient collagen.

Skin fibroblasts from two siblings with hydroxylysine-deficient collagen collagen (Ehlers-Danlos syndrome, type VI) contained normal levels of collagen prolyl hydroxylase activity but were markedly deficient in collagen lysyl hydroxylase activity. The deficiency was evident in all fractions of cell lysates, in low and high ionic strength buffers, and in detergent. Assays of mixtures of wild-type and mutant cell lysates indicated no activation of mutant enzyme by factors in wild-type cells or inhibition of normal enzyme by material in mutant cells. Wild type or mutant cells cultured with ascorbic acid (50 mug/ml of culture medium, added daily) contained approximately the same level of lysyl hydroxylase activity as cells cultured without ascorbate, but prolyl hydroxylase activity without ascorbate was depressed in both an average of 41%. The mutant lysyl hydroxylase was less stable at 37 degrees C than the wild type and did not form high molecular weight aggregates in low ionic strength buffers, as did the control enzyme. The activity of the mutant enzyme was maximally stimulated after dialysis against buffer solutions containing 10 mM dithiothreitol. When assayed in 100 muM dithiothreitol, the mutant enzyme exhibited a higher apparent Km for ascorbate (20 muM) than the wild type (4 muM). In 1.0 mM dithiothreitol the mutant enzyme's apparent Km for ascorbate was reduced to 5 muM. Wild type and mutant enzymes had the same apparent Km for alpha-keto-glutarate (20 muM). The properties of prolyl hydroxylase in wild type and mutant cells were identical: apparent Km's for ascorbate and alpha-ketoglutarate were 100 muM and 20 muM, respectively. If mutant enzyme protein with altered kinetic properties is the only enzyme functioning to hydroxylate lysyl residues in collagen, the variations in hydroxylysine content observed in collagen from different tissues in the subjects reported here could be in part due to differences in cofactor concentrations and in rate and sequence of events in collagen synthesis in different tissues.

Ehlers-Danlos syndrome in two siblings with deficient lysyl hydroxylase activity in cultured skin fibroblasts but only mild hydroxylysine deficit in skin.

Two siblings suffered from Ehlers-Danlos syndrome characterized by skin fragility, joint laxity and dermal hyperelasticity. The association with microcornea and muscle hypotonia allowed the preliminary classification into type VI according to McKusick. Ultrastructure analysis of skin biopsies revealed poor integration of collagen fibrils into fibres; accordingly, the texture of the connective tissue appeared irregular. Lysyl hydroxylase activity of cultured skin fibroblasts was markedly reduced in the cells of the two patients. Preliminary studies revealed intermediate activity in the cells cultured from the skin of the parents. This finding suggested an autosomal recessive mode of inheritance. Unexpectedly and in contrast to the 3 cases reported in the literature, the hydroxylysine deficit in the patients' skin was, for reasons not yet understood, only mild. Therefore, amino acid analysis of skin is not adequate for the diagnosis of lysyl hydroxylase-deficient Ehlers-Danlos syndrome type VI.

Hydroxylysine-deficient skin collagen in a patient with a form of the Ehlers-Danlos syndrome.

Two sibs with the Ehlers-Danlos syndrome, one of whom was shown to have hydroxylysine-deficient collagen, are described. In addition to the usual features of the Ehlers-Danlos syndrome (loose-jointedness and excessively stretchable, fragile, and bruisable skin), these patients had severe scoliosis and fragility of ocular tissues leading to rupture of the globe or retinal detachment. This combination of symptoms was tentatively classified as Ehlers-Danlos syndrome, Type VI. The condition is inherited as an autosomal recessive. The activity of lysyl hydroxylase was present at a reduced level in fibroblasts cultured from the patient's skin.