6.1. Lack of collagen type specificity for lysyl hydroxylase isoforms (I)

The mRNA expression levels of three LH isoforms (LH1, LH2, and LH3) have been studied in several different human cell lines. They were present in all human cells tested. There is a five-fold variation in the LH1 mRNA levels and a 20 to 30-fold variation in LH2 mRNA levels, whereas the variation for LH3 is only two-fold among different human cells. LH1 is the major isoform in skin fibroblasts, pancreas adenocarcinoma cells, and placenta trophoblastic tumor cells, being about 50% of the total lysyl hydroxylases. This explains the finding that the hydroxylysine content of collagens is most noticeably reduced in the skin when tissue samples are analyzed from patients with EDS VI, a disease caused by a LH1 gene defect (Hyland et al. 1992, Hautala et al. 1993, Ha et al. 1994, Heikkinen et al. 1997, Yeowell & Walker 1997, Brinckmann et al. 1998, Pajunen et al. 1998, Pousi et al. 1998, Heikkinen et al. 1999, Walker et al. 1999, Yeowell & Walker 1999b, Pousi et al. 2000, Yeowell et al. 2000a, Yeowell et al. 2000b). LH2 is the major form in fibrosarcoma, osteosarcoma, and hepatoblastoma cells, constituting about 60% of total LH in those cells. LH3 is a major form in kidney adenocarcinoma cells, about half of the LH within these cells being LH3. Todate no heritable disorders have been reported associated with a gene defect in LH2 and/or LH3.

The hydroxylysine content varies in the triple-helical regions of different collagen types. Type III and type I collagens contain only 6-10 hydroxylysyl residues per polypeptide chain, whereas type V and type IV contain a three- to five-fold higher amount of hydroxylysine (Kivirikko et al. 1992, Kielty et al. 1993). The initial suggestion for the existence of collagen type-specific LH isoforms was based on the findings that lysines in collagen type II, IV, V were hydroxylated normally in EDS VI patients (Ihme et al. 1984), and that the residual LH activity of EDS VI cells appears to preferentially hydroxylate lysines in type IV collagen (Risteli et al. 1980). The mRNA levels of LH isoforms and the α subunit of prolyl 4-hydroxylase, another enzyme involved in collagen biosynthesis were compared with the mRNAs of the most abundant collagen types (I, III, IV, V). The data do not indicate any significant correlation between individual collagen types and LH isoforms or the α subunit of prolyl 4-hydroxylase, arguing against collagen type specificity in the enzyme reactions. The amino acid sequences surrounding the individual lysyl residues, rather than collagen type, are probably more important for determining which lysyl hydroxylase isoform catalyzes the hydroxylation reaction. Prolyl 4-hydroxylase activity has been widely used to estimate the rate of total collagen synthesis in many experimental models and in patients with various diseases (Kivirikko et al. 1992, Kielty et al. 1993, Prockop & Kivirikko 1995). The mRNA levels of LH1, LH2, and the α subunit of prolyl 4-hydroxylase are significantly correlated with each other in various human cell lines, suggesting that LH1 and LH2, together with prolyl 4-hydroxylase are coregulated with total collagen synthesis. It is remarkable that the mRNA levels of LH3 do not correlate with those of LH1, LH2, and the α subunit of prolyl 4-hydroxylase, implying a difference in their substrates and in the regulation of their gene expression. According to the phylogenetic analysis, LH3 is the oldest form and the others result from more recent duplication events (Ruotsalainen et al. 1999). It is highly possible that LH3 acts on other types of sequences in vivo in addition to collagenous sequences.