Lysyl hydroxylase-2b directs collagen cross-linking pathways in MC3T3-E1 cells.
To elucidate the roles of LH2b in collagen cross-linking, MC3T3-E1 cell clones expressing higher (S) or lower (AS) levels of LH2b were established. Compared with controls, the collagen cross-linking pattern was shifted toward hydroxylysine-aldehyde (S clones)- or lysine-aldehyde (AS clones)-derived pathways. The data indicate that LH2b directs collagen cross-linking pathways through its action on telopeptidyl lysine residues.


BACKGROUND
Lysine (Lys) hydroxylation is a post-translational modification of collagen critical for cross-linking and glycosylation. Currently, three isoforms of lysyl hydroxylase (LH) have been identified, but their specific functions are still not well defined. Recently, we proposed that LH2 might modulate collagen cross-linking pattern through its action on Lys residues located in the telopeptide domains of collagen.


METHODS
To directly test this hypothesis, several MC3T3-E1 cell-derived clones expressing higher (sense [S]) or lower (antisense [AS]) levels of LH2b, the predominant form of LH2 in this cell line, were established and cultured for 2 weeks, and collagen cross-links and precursor aldehydes in the matrices were analyzed.


RESULTS
In S clones tested, the ratio of dihydroxylysinonorleucine (DHLNL) to hydroxylysinonorleucine (HLNL) was significantly higher than the average of controls (76% and 140% increase, respectively), and the level of pyridinoline (Pyr) was elevated (100% and 150% increase, respectively). In contrast, when MC3T3-E1 cells were transfected with a LH2b antisense construct (AS clones), the DHLNL/HLNL ratios were significantly lower than that of controls (56% and 73% decrease, respectively), and Pyr was not detected. Furthermore, significant amounts of an aldol-derived cross-link, dehydrohistidinohydroxymerodesmosine, were produced ( approximately 0.3 mol/mol of collagen) in AS clones.


CONCLUSIONS
The data clearly show a critical role of LH2b in determining collagen cross-linking pathways, most likely through its action on telopeptidyl Lys residues.
Pornprasertsuk
Suchaya
S
Dental Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7455, USA.
Duarte
Wagner R
WR
Mochida
Yoshiyuki
Y
Yamauchi
Mitsuo
M
D016475:3T3 Cells; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D003094:Collagen / Q000096:biosynthesis* / Q000737:chemistry; D016373:DNA, Antisense / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D003895:Desmosine / Q000031:analogs & derivatives* / Q000032:analysis; D004151:Dipeptides / Q000032:analysis; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000031:analogs & derivatives* / Q000032:analysis; D047468:Immunoprecipitation; D007527:Isoenzymes / Q000032:analysis / Q000235:genetics / Q000502:physiology; D008239:Lysine / Q000378:metabolism; D051379:Mice; D010006:Osteoblasts / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D000596:Amino Acids; D016373:DNA, Antisense; D018076:DNA, Complementary; D004151:Dipeptides; D007527:Isoenzymes; D003895:Desmosine; C007428:5,5'-dihydroxylysylnorleucine; C010991:delta-hydroxylysylnorleucine; D006639:Histidine; C002527:histidinohydroxymerodesmosine; C027622:dehydrohistidinohydroxymerodesmosine; D012313:RNA; C015484:pyridinoline; D003094:Collagen; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; D008239:Lysine