High-performance liquid chromatographic analysis of urinary hydroxylysyl glycosides as indicators of collagen turnover

Moro, Luigi; Modricky, C.; Stagni, N.; Vittur, Franco; Bernard, Bernedetto de

doi:10.1039/an9840901621

Cited by 51 publications

(9 citation statements)

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“…The collagenous proteins in the cells and media were collected and digested by highly purified collagenase (Sigma, St. Louis, MO, USA) as described elsewhere [25]. After being hydrolysed in 2M NaOH, the amount of galactosylhydroxylysine (GH) and glucosylgalactosylhy‐droxylysine (GGH) as fluorescent dansyl derivatives were measured by slightly modifying the high‐performance liquid chromatography (HPLC) method presented by Moro et al [30]. GH and GGH standards were kindly obtained from Professor R. Tenni, Italy.…”

Section: Methodsmentioning

confidence: 99%

The glycosyltransferase activities of lysyl hydroxylase 3 (LH3) in the extracellular space are important for cell growth and viability

Wang

Kovanen

Raudasoja

et al. 2009

J Cellular Molecular Medi

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Lysyl hydroxylase (LH) isoform 3 is a post-translational enzyme possessing LH, collagen galactosyltransferase (GT) and glucosyltransferase (GGT) activities. We have demonstrated that LH3 is found not only intracellularly, but also on the cell surface and in the extracellular space, suggesting additional functions for LH3. Here we show that the targeted disruption of LH3 by siRNA causes a marked reduction of both glycosyltransferase activities, and the overexpression of LH3 in HT-1080 cells increases hydroxylation of lysyl residues and the subsequent galactosylation and glucosylation of hydroxylysyl residues. These data confirm the multi-functionality of LH3 in cells. Furthermore, treatment of cells in culture medium with a LH3 N-terminal fragment affects the cell behaviour, rapidly leading to arrest of growth and further to lethality if the fragment is glycosyltransferase-deficient, and leading to stimulation of proliferation if the fragment contains LH3 glycosyltransferase activities. The effect is reversible, the cells recovering after removal of the glycosyltransferase-deficient fragment. The findings were confirmed by overexpressing the full-length LH3 in native or mutated forms in the cells. The data indicate that the increase in proliferation depends on the glycosyltransferase activity of LH3. The overexpression of a glycosyltransferase-deficient mutant or targeted disruption of LH3 by siRNA in cells results in abnormal cell morphology followed by cell death. Our data clearly indicate that the deficiency of LH3 glycosyltransferase activities, especially in the extracellular space, causes growth arrest revealing the importance of the glycosyltransferase activities of LH3 for cell growth and viability, and identifying LH3 as a potential target for medical applications, such as cancer therapy.

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Section: Methodsmentioning

confidence: 99%

The glycosyltransferase activities of lysyl hydroxylase 3 (LH3) in the extracellular space are important for cell growth and viability

Wang

Kovanen

Raudasoja

et al. 2009

J Cellular Molecular Medi

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“…Both components are released into the circulation during collagen degradation and may be measured in urine by HPLC after appropriate derivatization [96,97]. The intrinsic advantage of hydroxylysine over that of hydroxyproline as a marker of bone resorption is that the glycosylated forms are not metabolized and are not in¯uenced by dietary components [94,95].…”

Section: Hydroxylysine-glycosides (Hyl-glycosides)mentioning

confidence: 99%

Molecular Markers of Bone Turnover: Biochemical, Technical and Analytical Aspects

Seibel

2000

Osteoporosis International

151

114

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“…Hydroxylysine glycosides were measured as previously reported (9). Briefly: aliquots of hydrolyzed tissue were derivatized using dansyl chloride following the method of Gray (10), and 20 μΐ were injected into a reversed-phase Ultrasphere-ODS (C 18 of 5 μιη particles) HPLC column, of 250 X 4.6 mm i. d. The HPLC Beckman Gold Model was connected with a fluorimeter (Shimadzu RF551), using an excitation wavelength of 366 nm and an emission wavelength of 490 nm.…”

Section: Hydroxylysine Glycoside Measurementsmentioning

confidence: 99%

17β-Estradiol and Tamoxifen Prevent the Over-Glycosylation of Rat Trabecular Bone Collagen Induced by Ovariectomy

Moro¹,

Bettica²,

Romanello³

et al. 1997

CCLM

Self Cite

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Summary:We have recently demonstrated that ovariectomy in the rat causes over-glycosylation of collagen which is restricted to trabecular bone. In order to obtain further evidence, we studied whether estrogen or tamoxifen treatment prevented over-glycosylation of trabecular bone collagen. Forty one-hundred-day-old female rats were subjected to ovariectomy (n = 30) or sham-operation (n = 10). Starting the day of the operation, sham-operated rats were treated with vehicle, while ovariectomized rats were divided into three groups and treated with vehicle (n = 10), estrogen (n = 10) or tamoxifen (n = 10). Five rats from each group were sacrificed at 115 and 145 days of age. Femurs and tibiae were separated into cortical and trabecular bone, demineralized, hydrolyzed and analyzed by HPLC for hydroxylysine glycosides and hydroxyproline content. Hydroxylysine glycoside content was expressed as a molar ratio with hydroxyproline. The results can be summarized as follows: 1) cortical bone collagen glycosylation did not vary among the different groups;2) over-glycosylation of trabecular bone collagen observed in the ovariectomized rats was prevented by the administration of either 17ß-estradiol or tamoxifen.These data demonstrated that estrogens affect glycosylation of trabecular bone collagen.

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High-performance liquid chromatographic analysis of urinary hydroxylysyl glycosides as indicators of collagen turnover

Cited by 51 publications

References 0 publications

The glycosyltransferase activities of lysyl hydroxylase 3 (LH3) in the extracellular space are important for cell growth and viability

The glycosyltransferase activities of lysyl hydroxylase 3 (LH3) in the extracellular space are important for cell growth and viability

Molecular Markers of Bone Turnover: Biochemical, Technical and Analytical Aspects

17β-Estradiol and Tamoxifen Prevent the Over-Glycosylation of Rat Trabecular Bone Collagen Induced by Ovariectomy

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