Abstract Adenoviral gene transfer is a valuable tool in molecular biology research. In order to be an efficient and safe vector, adenovirus structure and infection mechanism as well as molecular biology of the used transgene need to be well studied. The aim of this study was to evaluate the role of adenovirus as a gene transfer vector from several perspectives. Adenovirus uses receptor-mediated endocytosis in order to enter the target cell. The effect of Rab5 GTPase on adenovirus entry and gene transfer efficiency was examined first. Next, adenovirus was used as an investigatory tool in the cardiovascular research, focused on clarifying the role of adrenomedullin (AM) in heart and vascular remodeling. Finally, a model of adenoviral gene transfer into skin fibroblasts was used. The role of Rab5 GTPase in the adenovirus endocytosis was examined in HeLa cells using Cy3-labeled adenovirus, and gene transfer efficiency using β-galactosidase encoding adenovirus. Rab5 increased both adenovirus uptake and gene transfer, whereas dominant negative Rab5S34N decreased both endocytosis and gene transfer. The data indicate that Rab5 is needed in mediating the adenovirus uptake into the target cell. In the rat heart, adenovirus-mediated AM gene transfer transiently improved systolic function both in vivo and in vitro. AM caused activation of translocation of protein kinases C ε and δ, whereas phosphorylation of p38 mitogen activated protein kinase was decreased in the left ventricle. AM significantly attenuated the development of angiotensin II-induced cardiac hypertrophy. In rats with myocardial infarction, AM enhanced dilatation of left ventricle and thinning of anterior wall. The role of AM in neointima formation was evaluated in rat artery after endothelial injury. Intravascular AM gene transfer decreased neointimal growth and increased neointimal myofibroblasts apoptosis. These results show that AM regulates left ventricular systolic function and remodeling in the heart, and plays a role in pathological vascular remodeling. Adenovirus-mediated lysyl hydroxylase (LH) gene transfer into skin fibroblasts of type VI Ehlers-Danlos syndrome patient and rat skin increased functional LH production, elevated LH activity, and human LH mRNA production both in vitro and in vivo. LH gene replacement therapy may thus lead to possibilities to improve skin wound healing in Ehlers-Danlos syndrome patients.

Abstract Lysyl hydroxylase (EC 1.14.11.4, procollagen-lysine 2-oxoglutarate 5-dioxygenase, PLOD) catalyses the formation of hydroxylysine in collagens and in the other collagen like proteins. Hydroxylysine participates in the formation of cross-links between collagen molecules and can bind to the carbohydrates, galactose and glucosylgalactose. Patients with the type VIA Ehlers-Danlos syndrome (EDS) have characteristically a deficiency in hydroxylysine of collagen in their skin that is caused by reduced activity of lysyl hydroxylase 1. In this work the mutations were studied in detail in four different Ehlers-Danlos VIA patients. The first patient characterized in this study had a duplication of seven exons in the lysyl hydroxylase gene 1. The mutation was caused by homologous recombination of two identical 44-nucleotide regions of Alu sequences in introns 9 and 16 in the gene. This study also suggests that uniparental isodisomy does not explain the homozygosity of the mutation. The second patient was found to have two mutations in the gene for lysyl hydroxylase 1 in a compound heterozygote state. The study resulted in the discovery of the first deletion mutation in the gene. The deletion was caused by an Alu-Alu recombination that removes about 3 kb from the gene including all the exon 17 sequences. The other mutation causes deletion of exon 16 from the mRNA. Deletion of the penultimate nucleotide of intron 15 destroys the consensus sequence of the intron/exon boundary and thus causes the deletion. The third patient was described to have a nonsense codon in exon 14 of one allele which causes a reduction in the amount of lysyl hydroxylase mRNA and leads to aberrant RNA splicing in the cell. The other allele was concluded to be operationally null. In the last work two novel null mutations were found in the gene for lysyl hydroxylase 1. The first was a one nucleotide deletion in the acceptor splice site of intron 4 and the other an insertion of a C nucleotide in exon 2. The abnormal alleles lead to markedly decreased lysyl hydroxylase mRNA levels. This work revealed many exon deleted splicing variants of lysyl hydroxylase mRNA which were first discovered in affected cells, but traces of similarly spliced mRNA species were also found in the cytoplasm of normal human skin fibroblasts. These data indicate that the splicing machinery of the cell is leaky. In this thesis, several types of stuctural mutations in the DNA were found to be responsible for lysyl hydroxylase deficiency in patients with type VIA variant of EDS. The different mechanisms causing these mutations were also studied in detail.

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 (D187-191) 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.

Abstract Lysyl hydroxylase (LH, EC 1.14.11.4) catalyzes the post-translational hydroxylation of lysyl residues in collagens and other proteins with collagenous domains. The hydroxylysyl residues participate in the formation of collagen cross-links, and some of the hydroxylysyl residues are further glycosylated. Three lysyl hydroxylase isoforms LH1, LH2 and LH3, encoded by three individual genes have been characterized and one isoform, LH3 is a multifunctional enzyme containing lysyl hydroxylase, collagen galactosyltransferase (GT, E.C. 2.4.1.50) and glucosyltransferase (GGT, E.C. 2.4.1.66) activities in vitro. In this thesis the genes for the mouse lysyl hydroxylases were each mapped to a different chromosome. In addition, the roles of the lysyl hydroxylase isoforms were characterized in mice by studying their expression during development and the distribution of LH2 and LH3 in adult mice. The results revealed a widespread expression of the mouse lysyl hydroxylases during embryonic development whereas LH2 and LH3 showed tissue- or cell-specific expression patterns in the adult. Alternative splicing of the gene for LH2 also showed developmental and tissue-specific regulation. The different functions of LH3 were studied in vivo by generating three different LH3 manipulated mouse lines. Analysis of the mouse lines revealed that LH3 has lysyl hydroxylase and glucosyltransferase activities in vivo, and that, in particular, the glucosyltransferase activity of LH3 is essential for normal development. The loss of glucosyltransferase activity caused disruption of basement membranes leading to embryonic lethality while the absence of lysyl hydroxylase activity led to ultrastructural alterations in muscle and basement membranes and disorganization of collagen fibrils. The disruption of basement membrane was due to an intracellular accumulation of unglycosylated type IV collagen, whereas the ultrastructural alterations were related to the abnormal aggregation and distribution of underglycosylated type VI collagen. The results demonstrate that hydroxylysine-linked glycosylations are critical for the secretion of type IV collagen and its assembly into basement membranes, and for the assembly and distribution of type VI collagen.

Abstract Lysyl hydroxylase (E.C. 1.14.11.4) catalyzes the formation of hydroxylysine in collagens and other proteins with collagenous domains. The resulting hydroxylysine residues participate in the formation of collagen crosslinks, and serve as attachment sites for carbohydrate units. They have been regarded as non-essential, since the absence of lysyl hydroxylase 1 activity is not lethal, although it leads to the kyphoscoliotic type of Ehlers-Danlos syndrome, and since recombinant collagens I and III lacking any hydroxylysine form native-type fibrils in vitro. A novel human lysyl hydroxylase isoenzyme, lysyl hydroxylase 3, was identified, cloned and characterized here. The novel isoenzyme was expressed as a recombinant protein in insect cells, and the protein was shown to catalyze hydroxylation of lysine residues in vitro. No differences were found in the catalytic properties between the recombinant lysyl hydroxylases 3 and 1. The human lysyl hydroxylase 3 gene was shown to be 11.6 kb in size and to contain 19 exons. The introns contain 15 full-length or partial Alu retroposons, which are known to be involved in most human gene rearrangements that occur by homologous recombination. The three recombinant human lysyl hydroxylase isoenzymes were isolated here for the first time as homogenous proteins. Limited proteolysis data suggested that the lysyl hydroxylase polypeptides might consist of at least three distinct domains, A-C. The N-terminal domain A was found to play no role in lysyl hydroxylase activity as a recombinant B-C polypeptide was a fully active hydroxylase. This work also confirmed that lysyl hydroxylase 3 has collagen glucosyltransferase activity as well as trace amounts of collagen galactosyltransferase activity. However, the levels of these activities were so low that their biological significance remains to be determined. In the last part of this work, lysyl hydroxylase 3 knock-out mice were produced and analyzed. The homozygous null embryos were found to die at a very early stage of development due to lack of type IV collagen in the basement membranes. The data demonstrated that hydroxylysine formed by lysyl hydroxylase 3 is essential for early mouse development and that lysyl hydroxylase 1 or 2 cannot compensate for the lack of its function.

Abstract Lysyl hydroxylase (LH) catalyzes the post-translational formation of hydroxylysines in collagens and collagenous proteins. Three lysyl hydroxylase isoforms, LH1, LH2 and LH3, have been identified from different species. In addition, LH2 has two alternatively spliced forms, LH2a and LH2b. The hydroxylysines have an important role in the formation of the intermolecular collagen crosslinks that stabilize the collagen fibrils. Some of the hydroxylysine residues are further glycosylated. In this thesis the substrate amino acid sequence specificities of the LH isoforms were analyzed using synthetic peptide substrates. The data did not indicate strict amino acid sequence specificity for the LH isoforms. However, there seemed to be a preference for some sequences to be bound and hydroxylated by a certain isoform. Galactosylhydroxylysyl glucosyltransferase (GGT) catalyzes the formation of glucosylgalactosylhydroxylysine. In this study, LH3 was shown to be a multifunctional enzyme, possessing LH and GGT activities. The DXD-like motif, characteristic of many glycosyltransferase families, and the conserved cysteine and leucine residues in the N-terminal part of the LH3 molecule were critical for the GGT activity, but not for the LH activity of the molecule. The GGT/LH3 protein level was found to be decreased in skin fibroblasts and in the culture media of cells collected from members of a Finnish epidermolysis bullosa simplex (EBS) family, which was earlier reported to have a deficiency of GGT activity. In this study, we showed that the reduction of enzyme activity is not due to a mutation or lower expression of the LH3 gene. Our data indicate that the decreased GGT/LH3 activity in cells has an effect on the deposition and organization of the key extracellular matrix components, collagen types VI and I and fibronectin, and these changes are transmitted to the cytoskeletal network. These findings underline LH3 as an important extracellular regulator.

Abstract Lysyl hydroxylases (E.C. 1.14.11.4, LHs) have three isoenzymes that are found in humans and mice, and they hydroxylate lysine residues in collagens and other proteins containing collagenous sequences. The hydroxylysines formed are crucial for the intermolecular collagen crosslinks that stabilise collagen fibres, thereby providing the stiffness and stability required by various tissues. In addition, hydroxylysines serve as attachment sites for carbohydrates, whose functions on collagen molecules are not completely understood yet. In humans, lack of LH1 causes Ehlers-Danlos syndrome (EDS) VIA, which is characterised, for example, by severe progressive kyphoscoliosis and muscular hypotonia with joint laxity. Mutations in the LH2 gene are associated with Bruck syndrome, which is characterised by fragile bones with congenital joint contractures. In the present work recombinant human lysyl hydroxylases were produced in insect cells and purified to homogeneity. Limited proteolysis revealed that LHs consist of at least three structural domains. The N-terminal domain plays no role in the lysyl hydroxylase activity, but instead, is responsible for the recently reported glucosyltransferase activity of LH3, and the galactosyltransferase activity reported here for the first time. The LH polypeptide lacking the N-terminal domain is a fully active LH with Km values identical to those of full-length enzyme. In addition, direct evidence is shown that LH2, but not LH1 or LH3, hydroxylates the telopeptide lysine residues of fibrillar collagens. All three recombinant LHs were able to hydroxylate the synthetic peptides representing the helical hydroxylation sites in types I and IV collagens, with some differences in the Vmax and Km values. In addition, all three LHs hydroxylated the collagenous domain of coexpressed type I procollagen chain to similar extend. In this study mouse lines lacking LH3 or LH1 were created and analysed. Unexpectedly, the LH3 null mice died during the embryonal period due to fragmentation of basement membranes. Type IV collagen, one of the major components in basement membranes, aggregates on its way to extracellular space and is absent from the basement membranes making them fragile. This is most probably caused by abnormal processing of type IV collagen due to decreased glucosyltransferase activity of the LH3 null embryos. The first mouse model for human EDS VIA is presented here. The LH1 null mice did not have kyphoscoliosis characteristic of EDS VIA, but showed gait abnormalities due to muscular hypotonia and possible joint laxity, as also seen in EDS VIA patients. In addition, the null mice died occasionally from aortic ruptures. Ultra structural analysis revealed degradation of smooth muscle cells and abnormal collagen fibres even in non-ruptured aortas of LH1 null mice. The hydroxylation of lysine residues and crosslinking in LH1 null mice were also abnormal, as in human EDS VIA patients. The LH1 null mouse line provides an excellent tool for analysing several aspects of human EDS VIA, including muscular hypotonia, abnormalities in collagen fibres and their crosslinking.

Abstract Collagens are subject to extensive post-translational modifications, including the formation of 4-hydroxyproline, 3-hydroxyproline and hydroxylysine. These reactions are catalyzed by collagen prolyl 4-hydroxylases (C-P4Hs), prolyl 3-hydroxylases (P3Hs) and lysine hydroxylases (LHs), which belong to the 2-oxoglutarate-dependent dioxygenase family and require oxygen for their reaction. 4-Hydroxyproline residues have for a long time been known to be required for the stability of the collagen triple helix, but the role of prolyl 3-hydroxylation was revealed only a few years ago when mutations in P3H1 and the consequent loss of a single 3-hydroxyproline in collagen I was shown to cause recessive osteogenesis imperfecta. In this thesis the human P3H isoenzymes were expressed as recombinant enzymes, and analyses of their tissue expression and kinetic properties revealed that P3H2 is located in tissues rich in basement membranes and that it hydroxylates collagen IV, the major basement membrane collagen. The roles of the collagen hydroxylases and collagen IV in basement membrane formation were further studied using Madin-Darby canine kidney (MDCK) epithelial cells as an in vitro model for cell polarization. 4-Hydroxyproline also has a pivotal role in the system of cellular response to reduced oxygen levels (hypoxia). At a normal oxygen concentration, two proline residues in the α subunit of the hypoxia-inducible factor (HIF) are 4-hydroxylated by the HIF-P4Hs, which target HIF-α for proteasomal degradation. In hypoxia, the HIF-P4Hs are inactive, and the α subunit thus escapes degradation, dimerizes with a β subunit and after recruiting transcriptional coactivators induces the transcription of hypoxia-responsive genes in order to adapt the cell to hypoxia. Three human HIF-α subunits have been characterized to date, of which the third is known to be subject to extensive alternative splicing, with one of the splicing variants acting as a negative regulator of the hypoxia responsive system. Four novel splicing variants generated from the human HIF-3α locus are characterized here, and the expression of HIF-3α variants has been shown to be upregulated by hypoxia in a HIF-1 dependent manner. Further studies on the binding partners and transcriptional activity of HIF-3α revealed that this subunit has a more complex role in the adaptation of cells to hypoxia than had been expected. / Tiivistelmä Kollageenit ovat valkuaisaineita, joihin kohdistuu useita synteesin jälkeisiä muokkauksia kuten 4-hydroksiproliinin, 3-hydroksiproliinin ja hydroksilysiinin muodostuminen. Näitä reaktioita katalysoivat kollageeniprolyyli-4-hydroksylaasit (C-P4H:t), prolyyli-3-hydroksylaasit (P3H:t) ja lysyylihydroksylaasit (LH:t), jotka kuuluvat 2-oksoglutaraattidioksygenaasien entsyymiperheeseen ja tarvitsevat happea reaktioonsa. 4-hydroksiproliinitähteiden on kauan tiedetty stabiloivan kollageeninrakenteen, kun taas 3-hydroksiproliinitähteiden merkitys on selvinnyt vasta viime vuosina. Mutaatiot P3H1-isoentsyymiä koodittavassa geenissä ja sen seurauksena yhden ainoan 3-hydroksiproliinitähteen puuttuminen kollageenissa I johtavat vaikeaan luustosairauteen, osteogenesis imperfectaan. Tässä väitöskirjassa ihmisen P3H:t tuotettiin rekombinanttiproteiineina. Tulokset paljastivat, että P3H2 ilmentyy erityisesti kudoksissa, joissa on paljon tyvikalvorakenteita ja että P3H2 hydroksyloi tehokkaasti kollageeni IV:n kaltaisia synteettisiä peptidejä. Lisäksi koiran munuaisten epiteelisoluihin pohjautuvaa in vitro-mallia käytettiin apuna tutkiessamme kollageeneja hydroksyloivien entsyymien ja kollageenin IV roolia tyvikalvon muodostumisessa sekä solujen polarisaatiossa. Kollageenia stabiloivan tehtävänsä lisäksi 4-hydroksiproliinilla on myös merkittävä rooli solujen vasteessa vähähappisille olosuhteille (hypoksia). Normaalissa happiosapaineessa (normoksia), hypoksiaindusoituvan tekijän (HIF) α-alayksikköön muodostuu HIF-P4H entsyymien katalysoimana kaksi 4-hydroksiproliinitähdettä, jotka kohdistavat α-alayksikön proteasomaaliseen hajotukseen. Hypoksiassa HIF-P4H:t eivät kykene toimimaan, jolloin α-alayksikkö säästyy hajotukselta, muodostaa kompleksin β-alayksikön kanssa ja sitoo transkriptiokofaktoreita. HIF-kompleksi kykenee tällöin lisäämään hypoksiassa tarvittavien geenien luentaa. Tänä päivänä tunnetaan kolme HIF α-alayksikköä, joista HIF-3α:sta tiedetään esiintyvän useita erilaisia silmukointimuotoja ja yhden näistä muodoista tiedetään toimivan negatiivisena säätelijänä hypoksiavasteessa. Tässä väitöskirjatyössä on tunnistettu neljä uutta HIF-3α:n silmukointimuotoa ja osoitettu että HIF-3α:n määrä kasvaa hypoksiassa HIF-1:n säätelemänä. Lisäksi sitoutumis- ja transkriptiokokeet paljastivat, että HIF-3α:n rooli hypoksiavasteessa on monimutkaisempi kuin aikaisemmin kuviteltiin.

Abstract The extracellular matrix is not just a scaffold for cells and tissues, but rather a dynamic part of the human body. Characteristics of collagens, the major protein components of the extracellular matrix, are determined already during synthesis and mutations in genes encoding collagens, unbalance of regulators or dysfunction of collagen modifying enzymes, for instance, can lead to severe clinical complications. Certain hydroxylysine residues formed by lysyl hydroxylases (LHs) function in collagens as precursors of collagen cross-links that stabilize collagenous structures and thereby tissues. In humans, a deficiency of LH1, which is known to hydroxylate lysines in the helical regions of collagen polypeptides, causes Ehlers-Danlos syndrome VIA (EDS VIA). It is characterized e.g. by progressive kyphoscoliosis and hypermobile joints. Mutations in LH2, which is known to hydroxylate lysines in the telopeptides of collagen polypeptides, cause Bruck syndrome type 2 (BS2). BS2 patients suffer from fragile bones and congenital joint contractures, for instance, but the syndrome is usually not lethal. In this work we have generated and analyzed genetically modified LH1 and LH2 null mouse lines to study the in vivo functions and roles of these enzymes. Analyses concentrated also on collagen cross-links that were determined from several null or heterozygous mouse tissues. In the present work we also studied the effects of known BS2 mutations on recombinant human LH2 polypeptides to understand the molecular pathology of the syndrome. As an animal model for human EDS VIA, LH1 null mice had certain characteristics typical for EDS VIA, such as muscular hypotonia, but generally the symptoms were milder. Like EDS VIA patients, the mice have an increased risk of arterial ruptures and ultrastructural changes can be seen in the wall of the aorta, explained by inadequate helical lysine hydroxylation accompanied by a changed cross-linking state of tissues. Similarly, analysis of the LH2 null mouse line demonstrated the importance of the enzyme in cross-link formation. We showed that even a reduced amount of LH2 in adult mice changes the cross-linking pattern in tissues and a total lack of the enzyme leads to embryonic lethality. Furthermore, we demonstrated that LH2 is particularly important in tissue structures supporting blood vessels in the developing mouse embryo or in extraembryonic tissues. Finally, our in vitro studies with recombinant human LH2 polypeptides revealed that the known BS2 mutations severely affect the activity of the enzyme thus explaining the clinical symptoms of the patients, but the mutations do not lead to a total inactivation of the enzyme, which may be critical for the survival of patients. / Tiivistelmä Solunulkoinen matriksi ei ole ainoastaan soluja ja kudoksia tukeva rakenne, vaan se on dynaaminen osa ihmiskehoa. Kollageenien, solunulkoisen matriksin yleisimpien proteiinien ominaisuudet määräytyvät jo kollageenien synteesivaiheessa ja mutaatiot kollageeneja koodittavissa geeneissä, säätelytekijöiden epätasapaino tai esimerkiksi kollageeneja muokkaavien entsyymien toimintahäiriöt voivat johtaa vaikeisiin kliinisiin komplikaatioihin. Tietyt lysyylihydroksylaasien (LH) muodostamat hydroksilysiinitähteet toimivat kollageeneissa kollageeniristisidosten esiasteina. Ristisidokset vakauttavat kollageenirakenteita ja siten myös kudoksia. LH1 hydroksyloi lysiinejä kollageenipolypeptidien kolmoiskierteisellä alueella ja ihmisellä entsyymin puutos aiheuttaa tyypin VIA Ehlers-Danlosin syndrooman (EDS VIA), jossa potilailla on esimerkiksi etenevää kyfoskolioosia ja yliliikkuvat nivelet. Mutaatiot LH2-entsyymissä, joka hydroksyloi lysiinejä kollageenipolypeptidien telopeptidialueilla, aiheuttavat tyypin 2 Bruckin syndrooman (BS2). BS2-potilaat kärsivät mm. luiden hauraudesta ja niveljäykkyydestä, mutta syndrooma ei yleensä ole letaali. Tässä työssä loimme ja analysoimme geneettisesti muunnellut LH1 ja LH2 hiirilinjat, joiden kyseinen LH-geeniaktiivisuus on hiljennetty. Linjojen avulla halusimme tutkia näiden entsyymien toimintaa ja merkitystä in vivo. Analyysit keskittyivät myös kollageeniristisidoksiin, joita tutkittiin useista poistogeenisten tai heterotsygoottisten hiirten kudoksista. Ymmärtääksemme BS2:n molekyylipatologiaa, tutkimme tässä työssä myös tunnettujen BS2-mutaatioiden vaikutuksia ihmisen LH2-rekombinanttiproteiinissa. EDS VIA:n eläinmallina LH1 poistogeenisillä hiirillä on joitakin ominaisuuksia, kuten lihashypotonia, jotka ovat tyypillisiä EDS VIA:lle, mutta yleisesti oireet ovat lievempiä. Kuten EDS VIA-potilailla, hiirillä on kohonnut valtimoiden repeytymisriski ja aortan seinämän ultrarakenteessa voidaankin havaita muutoksia. Oireita voidaan selittää riittämättömällä kollageenien kolmoiskierteisen alueen lysiinien hydroksylaatiolla, joka muuttaa kollageenien ristisidostilaa kudoksissa. Myös LH2-hiirilinjan analysointi osoitti kyseisen entsyymin tärkeyden ristisidosten muodostamisessa. Jo alentunut LH2:n määrä aikuisissa hiirissä muuttaa kudosten kollageeniristisidoksia ja täydellinen entsyymin puuttuminen johtaa sikiön kuolemaan. Lisäksi osoitimme, että LH2 on erityisen tärkeä kudosrakenteissa, jotka tukevat kehittyvän hiiren sikiön tai sikiön ulkopuolisten kudosten verisuonia. In vitro-tutkimukset ihmisen LH2-rekombinanttiproteiinilla paljastivat, että tunnetut BS2-mutaatiot vaikuttavat erittäin haitallisesti entsyymin toimintaan, mikä selittää potilaiden kliiniset oireet, mutta mutaatiot eivät kuitenkaan aiheuta entsyymin täydellistä inaktivaatiota, mikä voi olla kriittistä potilaiden selviytymisen kannalta.

Abstract Collagens and collagenous proteins undergo several post-translational modifications that are important for their structure and functions. Lysine hydroxylation produces hydroxylysines, which are important for collagen cross-link formation and provide attachment sites for galactose and glucosylgalactose. Glycosylated hydroxylysines are crucial for embryonic development and the assembly of certain collagen types. They may also facilitate interactions between collagen and adjacent molecules as well as control the diameter of collagen fibrils. Lysine hydroxylation is catalyzed by three lysyl hydroxylases (LH1, LH2 and LH3). In addition to lysyl hydroxylase activity, LH3 possesses collagen galactosyltransferase (GT) and glucosyltransferase (GGT) activities. In this study, polyclonal antibodies against the lysyl hydroxylase isoforms were produced for protein level studies to localize the expression and understand the functions of the different isoenzymes. The results indicated ubiquitous expression during embryonic development compared to the more restricted, cell and tissue specific expression patterns observed in adult mouse tissues. Differences were seen also in the alternative splicing of LH2 during embryogenesis and between tissue types. Analyses of the subcellular localization revealed that LH3 is also present in extracellular space. Tissue and cell specific differences were noted in the distribution of LH3 between cellular compartments. Substrate analysis suggested an additional and novel role for LH3 as an enzyme catalyzing lysine modifications of collagenous proteins in the extracellular space. The importance of LH1 and LH2 has been highlighted in Ehlers-Danlos type VI and Bruck syndromes, respectively. In this study, the lysyl hydroxylase 3 gene was linked to a heritable disorder for the first time. Urinary screening revealed a patient that lacked a glucosylgalactosyl derivative of a pyridinium cross-link. The GGT activity levels measured from the patient’s serum and lymphoblastoid cells were also reduced, which suggested a defect in the lysyl hydroxylase 3 gene. Genetic analyses revealed two mutations, one in each allele of LH3 in this compound heterozygous patient. Recombinant mutant proteins showed defects in lysyl hydroxylase and collagen glycosyltransferase activities, respectively. In conclusion, it was shown that a defect in LH3 catalyzed modifications leads to a novel disorder, which shares features with many other connective tissue disorders.