Chapter 7. Conclusions

The collagen type specificity of the LH isoforms in different human cell lines was first investigated in this study. The mRNA expression levels of the LH isoforms do not correlate with those of any major individual collagen type (I, III, IV, V), indicating a lack of collagen type specificity among the LH isoforms. The mRNA expression level of LH1, LH2 and the α-subunit of prolyl 4-hydroxylase showed a high correlation with each other whereas LH3 mRNA did not show any correlation to the mRNA levels of LH1, LH2 or the α-subunit of prolyl 4-hydroxylase. This suggests that LH3 may act on a different substrate in vivo. More studies are required to make clear the difference in substrate specificity of the LH isoforms.

It was also demonstrated in this study that the full length LH3 and C. elegans LH are multifunctional proteins, possessing LH, GT and GGT activities, which are able to catalyze three consecutive steps in building the unique hydroxylysine linked carbohydrate units in collagens. It reveals for the first time the cDNA sequence of GT and GGT, which enables us to search for the gene mutations in abnormal glycosylation-related diseases, although no heritable disorders related to this gene have been reported so far.

Subcellular localization of human LH3 was also studied in this work. It is located mainly in the ER whereas some is found in the Golgi complex probably due to overexpression, being similar to human LH1. This is in good agreement with the known intracellular sites of hydroxylation and glycosylation of lysyl residues in collagen biosynthesis. No corresponding data are available for human lysyl hydroxylase isoform 2 (LH2a and LH2b). The functions of human LH2a and LH2b remain to be elucidated.

The amino acids important for the catalytic activity of glycosyltransferases associated with human LH3 have been shown in this study to be localized at the amino-terminal part of the molecule, separate from the active site of LH. This enables us to design a knock out or transgenic study to manipulate LH and glycosyltransferase activities separately in mice to see if these activities associated with LH3 have the functions in vivo. Furthermore, the studies may lead to the elucidation of the functions of the glycosylation of hydroxylysyl residues in vivo.

The His-tagged constructs of the LH isoforms were expressed in E. coli or insect cells. Purification was carried out easily and conveniently by Nickel-NTA-Agarose chromatography. UDP-glucuronic acid affinity chromatography resulted in pure recombinant LH3/C. elegans LH protein. This approach will facilitate crystallization of human LH3 and C. elegans LH to study in detail the molecular structure of these multifunctional proteins.