Defective collagen crosslinking in bone, but not in ligament or cartilage, in Bruck syndrome: Indications for a bone-specific telopeptide lysyl hydroxylase on chromosome 17

Lysyl hydroxylase (LH) catalyzes the formation of hydroxylysine in collagens; three human isoenzymes have been cloned so far. We report here on the purification of all three recombinant isoenzymes to homogeneity from the medium of cultured insect cells, and we demonstrate that they are all homodimers. Limited proteolysis experiments identified two main protease-sensitive regions in the monomers of about 80 -85 kDa, corresponding to three fragments A-C (from the N to C terminus), with molecular masses of about 30, 37, and 16 kDa, respectively. Fragment A was found to play no role in LH activity as a recombinant B-C polypeptide constituted a fully active hydroxylase with K m values for cosubstrates and the peptide substrate that were identical to those of the full-length enzyme. LH3, but not LH1 and LH2, has also been reported recently (Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., and Myllylä , R. (2000) J. Biol. Chem. 275, 36158 -36163) to possess collagen glucosyltransferase activity. We confirm this highly surprising finding here and extend it by demonstrating that LH3 may also possess trace amounts of collagen galactosyltransferase activity. All the glucosyltransferase and galactosyltransferase activity of LH3 was found to reside in fragment A, which played no role in the hydroxylase activity of the polypeptide. This fragment is about 55% identical and 80% similar to the corresponding fragments of LH1 and LH2. However, the levels of the glycosyltransferase activities are so low that they may be of little biological significance. It is thus evident that human tissues must have additional glycosyltransferases that are responsible for most of the collagen glycosylation in vivo.

Section: Discussionmentioning

confidence: 99%

Characterization of Three Fragments That Constitute the Monomers of the Human Lysyl Hydroxylase Isoenzymes 1–3

Rautavuoma

Takaluoma

Passoja

et al. 2002

“…In skin, the telopeptides are not hydroxylated, and no such rearrangement of the Schiff base cross-link occurs. These two different chemistries, dependent on telopeptide lysine hydroxylation, lead to the formation of different mature cross-links by further reaction of the difunctional cross-links in a tissuespecific manner: hydroxylation of the telopeptide lysine residues appears to be accomplished by tissue-specific enzymes, distinct from those that hydroxylate lysines in the helix (2). For the hydroxylysyl aldehyde pathway (bone, cartilage, and tendon), the difunctional cross-links can combine with another hydroxylysine aldehyde-derived component to form a pyridinium cross-link.…”

mentioning

confidence: 99%

Structural Characterization of Pyrrolic Cross-links in Collagen Using a Biotinylated Ehrlich's Reagent

Brady¹,

Robins²

2001

Self Cite

The structures of pyrrolic forms of cross-links in collagen have been confirmed by reacting collagen peptides with a biotinylated Ehrlich's reagent. This reagent was synthesized by converting the cyano group of N-methyl-N-cyanoethyl-4-aminobenzaldehyde to a carboxylic acid, followed by conjugation with biotin pentylamine. Derivatization of peptides from bone collagen both stabilized the pyrroles and facilitated selective isolation of the pyrrole-containing peptides using a monomeric avidin column. Reactivity of the biotinylated reagent with collagen peptides was similar to that of the standard Ehrlich reagent, but heat denaturation of the tissue before enzyme digestion resulted in the loss of about 50% of the pyrrole cross-links. Identification of a series of peptides by mass spectrometry confirmed the presence of derivatized pyrrole structures combined with between 1 and 16 amino acid residues. Almost all of the pyrrole-containing peptides appeared to be derived from N-terminal telopeptide sequences, and the nonhydroxylated (lysine-derived) form predominated over pyrrole cross-links derived from helical hydroxylysine.The chemistry of the lysine-derived collagen cross-links is relatively complex, with collagen type, tissue location, and age of the protein all influencing the range of cross-links present (1). In bone, cartilage, and tendon, extensive hydroxylation of telopeptide lysine residues leads to the formation of hydroxylysyl aldehydes. These aldehydes interact with the ⑀ amino group of lysine or hydroxylysine residues from the adjacent helix to initially form Schiff bases that can chemically rearrange to form a more stable, keto-imine difunctional cross-link. In skin, the telopeptides are not hydroxylated, and no such rearrangement of the Schiff base cross-link occurs. These two different chemistries, dependent on telopeptide lysine hydroxylation, lead to the formation of different mature cross-links by further reaction of the difunctional cross-links in a tissuespecific manner: hydroxylation of the telopeptide lysine residues appears to be accomplished by tissue-specific enzymes, distinct from those that hydroxylate lysines in the helix (2). For the hydroxylysyl aldehyde pathway (bone, cartilage, and tendon), the difunctional cross-links can combine with another hydroxylysine aldehyde-derived component to form a pyridinium cross-link. These cross-links have been extensively characterized, and mechanisms of formation have been proposed (3, 4). However, many studies of cross-linked collagen peptides have implied the presence of other trifunctional cross-links because of a lack of stoichiometric amounts of the pyridinium compounds (5, 6). The possible presence of pyrrolic components in collagen arose from the observation that reaction of bone with p-dimethylaminobenzaldehyde gave a pink color characteristic of pyrroles (7); these reactive species were named Ehrlich chromogens. Later experiments used diazo-affinity columns (also consistent with a pyrrolic structure) to covalently bind the Ehrlich chromogen-c...

“…Analysis of bone samples from patients with this syndrome has demonstrated underhydroxylation of lysine residues in collagen telopeptides leading to aberrant cross-linking, while the lysines in the triple helical region are hydroxylated normally (20). No mutations in the human PLOD3 gene for LH3 have been characterized, but homozygous inactivation of the mouse Plod3 gene is lethal at E9.5 because of an abnormal aggregation of basement membrane-specific collagen IV (11,12).…”

mentioning

confidence: 99%

Tissue-specific Changes in the Hydroxylysine Content and Cross-links of Collagens and Alterations in Fibril Morphology in Lysyl Hydroxylase 1 Knock-out Mice

Takaluoma

Hyry

Lantto

et al. 2007

Self Cite

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.Lysyl hydroxylase (LH, 2 EC 1.14.11.4) catalyzes the hydroxylation of lysine residues mainly but not exclusively in -X-LysGly-triplets in collagens and proteins with collagen-like sequences (1). The enzyme resides within the endoplasmic reticulum and has three human and mouse isoenzymes, LHs 1-3 (1-7). The hydroxylysine residues formed have two important functions: they are essential for the stability of the intermolecular cross-links that provide the collagen fibrils with their tensile strength and mechanical stability, and they serve as attachment sites for carbohydrate units, either the monosaccharide galactose or the disaccharide glucosylgalactose (1). Collagen cross-link formation occurs in the extracellular matrix and is initiated by the conversion of specific lysine or hydroxylysine residues in the telopeptides, i.e. the short non-triple helical ends of collagen molecules, into the aldehydes allysine or hydroxyallysine, respectively, catalyzed by lysyl oxidase (8). The telopeptides are connected with the triple helical part of an adjacent molecule by difunctional immature cross-links in the characteristic staggered array of collagen molecules in a fibril. The three main fibril-forming collagens, types I, II, and III, have four cross-linking sites, one in each telopeptide and two in the triple helical region, close to its N-and C-terminal ends (9). If the residue in the telopeptide is hydroxyallysine, the difunctional cross-links can mature into trifunctional non-reducible cross-links comprising lysylpyri...