Human lysyl hydroxylase isoforms

Multifunctionality of human LH3 and the amino acids important for its collagen glycosyltransferase activities

Chunguang Wang

Biocenter Oulu, University of Oulu
Department of Biochemistry, University of Oulu

Abstract

Lysyl hydroxylase (EC1.14.11.4, LH) catalyzes post-translationally the hydroxylation of lysyl residues in collagens and other proteins with collagenous domains. Hydroxylysyl residues may also be glycosylated by hydroxylysyl galactosyltransferase (EC 2.4.1.50, GT) or galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66, GGT) to form galactosylhydroxylysyl or glucosylgalactosylhydroxylysyl residues, structures unique to collagen.

Three LH isoenzymes (LH1, LH2a/2b, LH3) have been characterized so far. We analyzed mRNA levels of these isoforms, as well as the mRNAs of the main collagen types (I, III, IV, V) and the α subunit of PH-4 in different human cell lines. Large variations were found in mRNA expression of LH1 and LH2 but not LH3. The mRNA levels of LH1, LH2, and the α subunit of PH-4 showed significant correlation with each other whereas LH3 correlated with none. No correlation was observed between the LH isoforms and individual collagen types.

Three human LH isoforms were expressed in different expression systems. The purified recombinant protein produced by LH3 cDNA was found to be the only one possessing LH, GT and GGT activities. The molecular weight of the partially purified LH3 expressed in Sf9 or Cos-7 cells corresponded to about 85 kDa whereas that in E. coli cells was about 81 kDa probably due to a deficiency of glycosylation in bacterial cells. The recombinant protein of C. elegans LH cDNA was expressed in a cell-free translation system and in E. coli cells. The data indicated that the glycosyltransferase activities, GT and GGT, were also associated with this gene product.

The sequence alignment of LH isoforms from different species revealed that there are 29 amino acids conserved between human LH3, mouse LH3 and C. elegans LH sequences and scattered evenly in the molecule, but differing from those of LH1 and LH2. In vitro mutagenesis data showed that the amino acids important for the glycosyltransferase activities were located at the amino-terminal part of the molecule, being separate from the LH active site. Mutation of a conserved LH3 specific, non-disulfide linked cysteine to isoleucine caused a dramatic reduction in GT and GGT activity but had no effect on LH activity. Mutations of the amino-terminal DxD motif (D_187-191) characteristic of many glycosyltransferases eliminated both GT and GGT activities, showing the importance of this motif for collagen glycosyltransferases and suggesting that it might serve as the Mn2+ binding site in the molecule.

Table of Contents

Acknowledgements

Abbreviations

List of original articles

1. Introduction

2. Review of literature

2.1. Collagens

2.1.1. Structure of collagen triple helix
2.1.2. Collagen types
2.1.3. Collagen biosynthesis

2.2. Hydroxylysine

2.2.1. Hydroxylysine in collagens and other proteins
2.2.2. Glycosylation of hydroxylysine in collagens and other proteins
2.2.3. Crosslinks in collagen

2.3. Lysyl hydroxylases

2.3.1. Molecular properties of lysyl hydroxylase
2.3.2. Reaction catalyzed by lysyl hydroxylase
2.3.3. Lysyl hydroxylase isoforms
2.3.4. Subcellular localization and distribution of LH isoforms in tissues
2.3.5. Ehlers-Danlos syndrome type VI

2.4. Collagen glycosyltransferases

2.4.1. Purification and molecular properties of collagen glycosyltransferases
2.4.2. Catalytic properties of collagen glycosyltransferases
2.4.3. Intracellular sites of collagen glycosylations
2.4.4. Collagen glycosyltransferase activities in physiological, pathological, and experimental conditions

3. Aims of the present work

4. Materials and Methods

4.1. Cell culture and transfection (I, II)

4.2. RNA isolation and Northern blot analysis (I)

4.3. Production of antibodies (I, II)

4.4. Microscopical study (II)

4.5. Immunoprecipitation (II)

4.6. Immunoblotting (I, II, III, IV)

4.7. In vitro translation (II, III, IV)

4.8. Site-directed in vitro mutagenesis (II, III, IV)

4.9. Expression and purification of the recombinant LH3 proteins (II, III, IV)

4.9.1. Constructs
4.9.2. Protein expression
4.9.3. Purification of His-tag protein by Nickel-NTA-Agarose
4.9.4. UDP-glucuronic acid affinity chromatography

4.10. GT and GGT activity measurements (II, III, IV)

4.11. Software (I, III)

5.1. Messenger RNA expression levels of LH isoforms and major collagen types I, III, IV, V in different human cell lines (I)

5.2. Characterization of the multifunctionality of human LH3 and C. elegans LH (II, III, IV)

5.2.1. Human LH3 is a multifunctional protein possessing GGT activity (II)
5.2.2. Human LH3 is the only isoenzyme possessing GT activity (IV)
5.2.3. GT and GGT activities are associated with C. elegans LH (III, IV)

5.3. The intracellular distribution of human LH3 (II)

5.4. Identification of the amino acids important for catalytic activities of GT and GGT associated with human LH3 and C. elegans LH (III, IV)

5.4.1. Active sites of GGT in human LH3 and C. elegans LH (III)
5.4.2. Active sites of GT in human LH3 (IV)

6. Discussion

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

6.2. Characterization of GT and GGT activities associated with human LH3 and C. elegans LH (II, III, IV)

6.2.1. Human LH3 is a multifunctional protein possessing LH, GT and GGT activities (II, IV)
6.2.2. C. elegans LH is multifunctional possessing also glycosyltransferase activities (III, IV)

6.3. Identification of the amino acids important for the glycosyltransferase activities associated with human LH3 and C. elegans LH (III, IV)

6.3.1. Active sites of GGT in human LH3 and C. elegans LH (III)
6.3.2. Active sites of GT in human LH3 (IV)

7. Conclusions

List of Tables
1. General steps in collagen biosynthesis and the post-translational processing enzymes

List of Figures
1. Biosynthesis of a fibrillar collagen. Procollagen polypeptide chains are synthesized on the ribosomes of the rough ER and secreted into the lumen, where the chains are modified by hydroxylation of certain prolyl and lysyl residues and glycosylation before chain association and triple helix formation. The procollagen molecules are secreted into the extracellular space where the N and C propeptides are cleaved by specific proteases. The collagen molecules then assemble into fibrils that are stabilized by the formation of crosslinks (Modified from Myllyharju and Kivirikko 2001 with permission of Taylor & Francis AB).
2. Hydroxylation reaction catalyzed by lysyl hydroxylase. One atom of oxygen is incorporated into the hydroxyl group of the lysyl residue in the peptide substrate and another one into 2-oxoglutarate, which is decarboxylated to form succinate and liberates CO₂.
3. The structure of the collagen-specific carbohydrate Glc-Gal-Hyl in peptide linkage. The carbohydrate unit may also consist only of the Gal portion (Modified from Kivirikko & Myllylä 1979 with permission of Academic Press).
4. Glycosylation reactions catalyzed by peptidyl GT and GGT (Modified from Kivirikko & Myllylä 1984 with permission of Elsevier Science).
5. Schematic representation of the mechanism proposed for the GGT reaction. At high Mn2+ concentrations, GGT probably binds two Mn2+ ions successively before binding to UDP-glucose. Due to the high Kd value at site II, this situation may not occur in vivo and therefore is not shown. Mn2+ need not to leave the enzyme between the catalytic cycles. GGT: galactosylhydroxylysyl glucosyltransferase; UDPglc: UDP-glucose; Glc-pept: glucosylated peptide (Modified from Kivirikko & Myllylä 1984 with permission of Elsevier Science).
6. Schematic representation of the events in collagen biosynthesis that occur within the cisternae of the rough ER. The propeptide is removed during the translocation across the membrane and hydroxylation of lysyl residues and glycosylation of hydroxylysyl residues are initiated while the polypeptide chains are still being assembled on the ribosomes. The reactions continue after the synthesis of complete pro α-chains until the triple helix is formed. LH: lysyl hydroxylase; GT: hydroxylysyl galactosyltransferase; GGT: galactosylhydroxylysyl glucosyltransferase; Gal: galactose; Glu: glucose (Modified from Kivirikko & Myllylä 1984 with permission of Elsevier Science).

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