Molecular biology of lysyl hydroxylases and collagen glycosyltransferases

Project leader: Raili Myllylä, Professor

Department of Biochemistry, University of Oulu, P.O. Box 3000, FI-90014 University of Oulu, Finland, e-mail: raili.myllyla (at) oulu.fi

Background and Significance of the Research

Collagens are a family of extracellular proteins that play an important role in maintenance of the structural integrity of tissues. Furthermore, the collagens, like other proteins, bind to growth factors as well as to other regulatory components of cells and modulate cellular metabolism and regulate cellular behaviour.

Collagen molecules are composed of three polypeptide chains, which fold to form a characteristic triple-helical structure. The biosynthesis of collagen requires a series of post-translational modifications and many of these are unique to collagens and other proteins with a collagen-like amino acid structure. The modifications, which occur within the rough endoplasmic reticulum (ER) of the cell, include hydroxylation of lysyl residues and glycosylation of hydroxylysyl residues. This glycosylation is unique to collagenous structures and the carbohydrate group can be either the monosaccharide galactose or the disaccharide glucosylgalactose. The side chains of the modified lysyl residues extend outward from the helix and have a high potential for lateral interactions between collagen molecules and other extracellular proteins. Modifications of lysyl residues are essential for the proper function of collagen, although the structural and regulatory roles of hydroxylysyl residues and glycosylated hydroxylysyl residues are not well understood. The focus of the research project is to clarify the functions of the hydroxylation of lysyl and glycosylation of hydroxylysyl residues of collagens, modifications unique to collagenous sequences.

Recent Progress

Our studies concern lysyl hydroxylase (LH) isoforms, recently focusing particularly on LH3 and LH2. We were the first group to characterize LH2 and LH3 (Valtavaara et al., J Biol Chem 272:6831, 1997; Valtavaara et al., J Biol Chem 273:12881, 1998). Our phylogenetic analyses indicate that LH3 is the oldest isoform among lysyl hydroxylase isoforms (Ruotsalainen et al., Matrix Biol 18:325, 1999). As recently indicated by our group (Heikkinen et al., J Biol Chem 275:36158, 2000; Wang et al., J Biol Chem 277:18568, 2002; Wang et al., Matrix Biol 21:559, 2002; Ruotsalainen et al., J Cell Sci 119:625-635, 2006), LH3 is a multifunctional enzyme possessing LH, collagen glucosyltransferase (GGT) and galactosyltransferase (GT) activities. We have identified the amino acids important for GGT and GT activities. Our data indicate that the active site is separate from the carboxy-terminal LH active site, the amino acids important for glycosyltransferase activities being located at the amino-terminal part of the molecule, having a DXD-like motif characteristic of many glycosyltransferases.

We have generated three different mouse lines for LH3. Mice with a mutation that blocked only the LH activity of LH3 developed normally, but showed defects in the structure of the basement membrane and in collagen fibril organization in newborn skin and lung. Analysis of a hypomorphic LH3 mouse line with the same mutation, however, demonstrated that reduction of the GGT activity of LH3 disrupts the localization of type IV collagen, and thus the formation of basement membranes during mouse embryogenesis, leading to lethality at embryonic day E9.5–14.5. Strikingly, survival of hypomorphic embryos and formation of the basement membrane were directly correlated with the level of GGT activity. In addition, an LH3-knockout mouse lacked GGT activity, leading to lethality at E9.5. The results confirm that LH3 has LH and GGT activities in vivo, LH3 is the main molecule responsible for GGT activity, and the GGT activity, not the LH activity of LH3, is essential for formation of the basement membrane. Together, our results demonstrate for the first time the importance of hydroxylysine-linked glycosylation for collagens.

We have evidence furthermore, that LH3, in addition to being an ER resident, is secreted from cultured cells and is found both in the medium and on cell surfaces associated with collagens or other proteins with collagenous sequences. In addition, LH3 is present in serum. LH3 in vivo is predominantly located in two compartments, in the ER and in the extracellular space, and the partitioning varies with tissue type. In mouse kidney the enzyme is located mainly intracellularly, whereas in mouse liver it is located solely in the extracellular space. Our data on LH2 indicate that it is located solely in the ER, in contrast to LH3. The extracellular localization and the ability of LH3 to modify lysyl residues of extracellular proteins in their native, non-denatured conformation reveal a new dynamic in extracellular matrix remodelling, suggesting a novel mechanism for adjusting the amount of hydroxylysine and hydroxylysine-linked carbohydrates in collagenous proteins.

Future Goals

This study is designed to further our understanding of the modifications of lysyl residues in collagens and a few other proteins with collagenous sequences. The project is focused on the molecular and cellular biology of enzymes modifying lysyl residues in collagens, i.e. lysyl hydroxylase, galactosyltransferase and glucosyltransferase. The study has the following goals:

1. to characterize the enzymes in more detail at the protein level
2. to characterize the substrate-specificity of the enzymes
3. to search for compounds that modulate enzyme activities
4. to search for mutations in the genes of the enzymes
5. to determine the function of the enzymes by generating transgenic animals and studying the consequences of such manipulations
6. to study the extracellular function of LH3

Publications from the subject

Salo, A., Wang, C., Sipilä, L., Sormunen, R, Vapola, M., Kervinen, P., Ruotsalainen, H., Heikkinen, J., & Myllylä, R.: Lysyl hydroxylase 3 (LH3) modifies proteins in the extracellular space, a novel mechanism for matrix remodelling. J Cell Physiol. 207, 644-653, 2006.

Ruotsalainen, H., Sipilä, L., Vapola, M., Sormunen, R., Salo, A., Uitto, L., Mercer, D.K., Robins, S.P., Risteli, M., Aszodi, A., Fässler, R. & Myllylä, R. Glycosylation catalyzed by lysyl hydroxylase 3 (LH3) is essential for basement membranes. J Cell Sci 119, 625-634, 2006.

Salo, A., Sipilä, L., Sormunen, R., Ruotsalainen, H., Vainio, S., & Myllylä, R.: The lysyl hydroxylase isoforms are widely expressed during mouse embryogenesis, but obtain tissue- and cell specific pattern in the adult. Matrix Biol. 25, 475-483, 2006.

Myllylä, R., Wang, C., Heikkinen, J., Juffer, A., Lampela, O., Risteli, M., Ruotsalainen, H., Salo, A., & Sipilä, L.: Expanding the lysyl hydroxylase toolbox: New insights into the localization and activities of lysyl hydroxylase 3 (LH3). J Cell Physiol. 212, 323-329, 2007

Myllylä, R., Wang, C., Heikkinen, J., Juffer, A., Lampela, O., Risteli, M., Ruotsalainen, H., Salo, A., & Sipilä, L.: Expanding the lysyl hydroxylase toolbox: New insights into the localization and activities of lysyl hydroxylase 3 (LH3). J Cell Physiol, 212, 323-329, 2007

Sipilä, L., Ruotsalainen, H., Sormunen, R., Baker, N.L., Lamande, S.R., Vapola, M., Wang, C., Sado, Y., Aszodi, A, Myllylä, R.: Secretion and assembly of type IV and VI collagens depend on glycosylation of hydroxylysines. J. Biol. Chem, 282, 33381-8, 2007

Salo, A., Cox, H., Farndon, P., Moss, C., Grindulis, H., Risteli, M., Robins, SP, Myllylä, R.: A connective tissue disorder caused by mutations of the lysyl hydroxylase 3 gene (PLOD3). Am. J. Hum. Genet., 83, 495-503, 2008.

Wang, C., Kovanen, V., Raudasoja, P., Eskelinen, S., Pospiech, H., Myllylä, R.: Glycosyltransferase activities of multifunctional lysyl hydroxylase 3 (LH3) in extracellular space are important for cell growth and viability. J Cell Mol Med 13, 508-521, 2009.

Risteli, M., Ruotsalainen, H., Salo, MA, Sormunen, R., Sipilä, L., Baker, NL, Lamande,
SR, Vimpari-Kauppinen, L., Myllylä, R.: Reduction of lysyl hydroxylase 3 causes deleterious changes in the deposition and organization of extracellular matrix. J. Biol. Chem., 284, 28204-28211, 2009.

Research group members:

• Raili Myllylä, Ph.D., Professor, emer
  
• Chunguang Wang, Ph.D.
  
• Heli Ruotsalainen, Ph.D.
  
• Jari Heikkinen, Ph.D.
  
• Antti Salo, Ph. D.
  
• Maija Risteli, Ph.D.
  
• Pia Mäkelä, a technician