Lysyl hydroxylase 3 (LH3) modifies proteins in the extracellular space, a novel mechanism for matrix remodeling

Lysyl hydroxylase 3 (LH3), the multifunctional enzyme associated with collagen biosynthesis that possesses lysyl hydroxylase and collagen glycosyltransferase activities, has been characterized in the extracellular space in this study. Lysine modifications are known to occur in the endoplasmic reticulum (ER) prior to collagen triple-helix formation, but in this study we show that LH3 is also present and active in the extracellular space. Studies with in vitro cultured cells indicate that LH3, in addition to being an ER resident, is secreted from the cells and is found both in the medium and on the cell surface associated with collagens or other proteins with collagenous sequences. Furthermore, in vivo, LH3 is present in serum. LH3 protein levels correlate with the galactosylhydroxylysine glucosyltransferase (GGT) activity of mouse tissues. This, together with other data, indicates that LH3 is responsible for GGT activity in the tissues and that GGT activity assays can be used to quantify LH3 in tissues. LH3 in vivo is 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. The extracellular localization and the ability of LH3 to modify lysyl residues of extracellular proteins in their native, nondenaturated conformation reveals a new dynamic in extracellular matrix remodeling, suggesting a novel mechanism for adjusting the amount of hydroxylysine and hydroxylysine-linked carbohydrates in collagenous proteins.     

Expanding the lysyl hydroxylase toolbox: new insights into the localization and activities of lysyl hydroxylase 3 (LH3)

Hydroxylysine and its glycosylated forms, galactosylhydroxylysine and glucosylgalactosylhydroxylysine, are post-translational modifications unique to collagenous sequences. They are found in collagens and in many proteins having a collagenous domain in their structure. Since the last published reviews, significant new data have accumulated regarding these modifications. One of the lysyl hydroxylase isoforms, lysyl hydroxylase 3 (LH3), has been shown to possess three catalytic activities required sequentially to produce hydroxylysine and its glycosylated forms, that is, the lysyl hydroxylase (LH), galactosyltransferase (GT), and glucosyltransferase (GGT) activities. Studies on mouse models have revealed the importance of these different activities of LH3 in vivo. LH3 is the main molecule responsible for GGT activity in mouse embryos. A lack of this activity causes intracellular accumulation of type IV collagen, which disrupts the formation of basement membranes (BMs) during mouse embryogenesis and leads to embryonic lethality. The specific inactivation of the LH activity of LH3 causes minor alterations in the structure of the BM and collagen fibril organization, but does not affect the lifespan of mutated mice. Recent data from zebrafish demonstrate that growth cone migration depends critically on the LH3 glycosyltransferase domain. LH3 is located in the ER loosely associated with the membranes, but, unlike the other isoforms, LH3 is also found in the extracellular space in some tissues. LH3 is able to adjust the amount of hydroxylysine and hydroxylysine-linked carbohydrates of extracellular proteins in their native conformation, suggesting that it may have a role in matrix remodeling.     

The glycosyltransferase activities of lysyl hydroxylase 3 (LH3) in the extracellular space are important for cell growth and viability

Lysyl hydroxylase (LH) isoform 3 is a post-translational enzyme possessing LH, collagen galactosyltransferase (GT) and glucosyltransferase (GGT) activities. We have demonstrated that LH3 is found not only intracellularly, but also on the cell surface and in the extracellular space, suggesting additional functions for LH3. Here we show that the targeted disruption of LH3 by siRNA causes a marked reduction of both glycosyltransferase activities, and the overexpression of LH3 in HT-1080 cells increases hydroxylation of lysyl residues and the subsequent galactosylation and glucosylation of hydroxylysyl residues. These data confirm the multi-functionality of LH3 in cells. Furthermore, treatment of cells in culture medium with a LH3 N-terminal fragment affects the cell behaviour, rapidly leading to arrest of growth and further to lethality if the fragment is glycosyltransferase-deficient, and leading to stimulation of proliferation if the fragment contains LH3 glycosyltransferase activities. The effect is reversible, the cells recovering after removal of the glycosyltransferase-deficient fragment. The findings were confirmed by overexpressing the full-length LH3 in native or mutated forms in the cells. The data indicate that the increase in proliferation depends on the glycosyltransferase activity of LH3. The overexpression of a glycosyltransferase-deficient mutant or targeted disruption of LH3 by siRNA in cells results in abnormal cell morphology followed by cell death. Our data clearly indicate that the deficiency of LH3 glycosyltransferase activities, especially in the extracellular space, causes growth arrest revealing the importance of the glycosyltransferase activities of LH3 for cell growth and viability, and identifying LH3 as a potential target for medical applications, such as cancer therapy.     

Lysyl hydroxylase 3 is a multifunctional protein possessing collagen glucosyltransferase activity

Lysyl hydroxylase (EC ) and glucosyltransferase (EC ) are enzymes involved in post-translational modifications during collagen biosynthesis. We reveal in this paper that the protein produced by the cDNA for human lysyl hydroxylase isoform 3 (LH3) has both lysyl hydroxylase and glucosyltransferase (GGT) activities. The other known lysyl hydroxylase isoforms, LH1, LH2a, and LH2b, have no GGT activity. Furthermore, antibodies recognizing the amino acid sequence of human LH3 and those against a highly purified chicken GGT partially inhibited the GGT activity. Similarly, a partial inhibition was observed when these antibodies were tested against GGT extracted from human skin fibroblasts. In vitro mutagenesis experiments demonstrate that the amino acids involved in the GGT active site differ from those required for LH3 activity.     

Identification of amino acids important for the catalytic activity of the collagen glucosyltransferase associated with the multifunctional lysyl hydroxylase 3 (LH3)

Collagen glucosyltransferase (GGT) activity has recently been shown to be associated with human lysyl hydroxylase (LH) isoform 3 (LH3) (Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., Myllyl?, R. (2000) J. Biol. Chem. 275, 36158-36163). The LH and GGT activities of the multifunctional LH3 protein modify lysyl residues in collagens posttranslationally to form hydroxylysyl and glucosylgalactosyl hydroxylysyl residues respectively. We now report that in the nematode, Caenorhabditis elegans, where only one ortholog is found for lysyl hydroxylase, the LH and GGT activities are also associated with the same gene product. The aim of the present studies is the identification of amino acids important for the catalytic activity of GGT. Our data indicate that the GGT active site is separate from the carboxyl-terminal LH active site of human LH3, the amino acids important for the GGT activity being located at the amino-terminal part of the molecule. Site-directed mutagenesis of a conserved cysteine at position 144 to isoleucine and a leucine at position 208 to isoleucine caused a marked reduction in GGT activity. These amino acids were conserved in C. elegans LH and mammalian LH3, but not in LH1 or LH2, which lack GGT activity. The data also reveal a DXD-like motif in LH3 characteristic of many glycosyltransferases and the mutagenesis of aspartates of this motif eliminated the GGT activity. Reduction in GGT activity was not accompanied by a change in the LH activity of the molecule. Thus GGT activity can be manipulated independently of LH activity in LH3. These data provide the information needed to design knock-out studies for investigation of the function of glucosylgalactosyl hydroxylysyl residues of collagens in vivo.     

Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture

Lysyl hydroxylase 3 (LH3), encoded by Plod3, is the multifunctional collagen-modifying enzyme possessing LH, hydroxylysine galactosyltransferase (GT), and galactosylhydroxylysine-glucosyltransferase (GGT) activities. Although an alteration in type I collagen glycosylation has been implicated in several osteogenic disorders, the role of LH3 in bone physiology has never been investigated. To elucidate the function of LH3 in bone type I collagen modifications, we used a short hairpin RNA technology in a mouse osteoblastic cell line, MC3T3-E1; generated single cell-derived clones stably suppressing LH3 (short hairpin (Sh) clones); and characterized the phenotype. Plod3 expression and the LH3 protein levels in the Sh clones were significantly suppressed when compared with the controls, MC3T3-E1, and the clone transfected with an empty vector. In comparison with controls, type I collagen synthesized by Sh clones (Sh collagen) showed a significant decrease in the extent of glucosylgalactosylhydroxylysine with a concomitant increase of galactosylhydroxylysine, whereas the total number of hydroxylysine residues was essentially unchanged. In an in vitro fibrillogenesis assay, Sh collagen showed accelerated fibrillogenesis compared with the controls. In addition, when recombinant LH3-V5/His protein was generated in 293 cells and subjected to GGT/GT activity assay, it showed GGT but not GT activity against denatured type I collagen. The results from this study clearly indicate that the major function of LH3 in osteoblasts is to glucosylate galactosylhydroxylysine residues in type I collagen and that an impairment of this LH3 function significantly affects type I collagen fibrillogenesis.     

Golgi and secreted galactosyltransferase

Galactosyltransferase (GT) belongs to the glycosyltransferases. In several tissues and cell lines, the enzyme is localized by immunocytochemistry to the two to three trans cisternae of the Golgi complex and may thus be considered a specific membrane component of this type of endomembrane. As a consequence, it is the most common Golgi "marker" enzyme in cell fractionation studies. Study of its biosynthesis, membrane orientation, and turnover in several tissues and cultured cell lines has broadened our knowledge about Golgi function itself. The enzyme is oriented towards the lumen of the cisternal space. In this orientation, it catalyzes the transfer of galactose to glycoprotein-bound acetylglucosamine and, in the presence of alpha-lactalbumin, to glucose, as shown in the Golgi complex of mammary gland epithelial cells. The enzymatic properties of GT are well known. The metabolism of GT has been extensively studied in HeLa and human hepatoma cells. The enzyme is synthesized in the rough endoplasmic reticulum (RER) and provided with one N-linked oligosaccharide and palmitate residues. In the Golgi complex, terminal sugars are attached to the N-linked oligosaccharide and extensive O-glycosylation takes place. The half-life of the enzyme is about 20 hr, after which a soluble form appears in the culture medium. Release of GT into the medium is observed in all cell lines studied. This phenomenon is in accordance with the presence of soluble GT in body fluids such as serum, ascites, milk, and saliva. In patients suffering from ovarian and breast cancer, increased levels of GT enzyme activity have been reported. Whether extracellular GT is of biological significance is still a point of discussion.     

Involvement of a cysteine protease in the secretion process of human xylosyltransferase I

Xylosylation of core proteins takes place in the Golgi-apparatus as the transfer of xylose from UDP-xylose to specific serine residues in proteoglycan core proteins. This initial and rate-limiting step in glycosaminoglycan biosynthesis is catalyzed by human xylosyltransferase I (XT-I). XT-I is proteolytically cleaved from the Golgi surface and shed in its active form into the extracellular space. The secreted, circulating glycosyltransferase represents a serum biomarker for various diseases with an altered proteoglycan metabolism, whereas a physiological function of secreted XT-I is still unknown. To shed light on the secretion process of XT-I and on its biological function, the cleavage site was examined and the group of proteases involved in the cleavage was identified in this study. The peptide mass fingerprint from partly purified secreted XT-I revealed the cleavage site to be localized in the aminoterminal 231 amino acids. The addition of a cysteine protease inhibitor cocktail to cells recombinantly expressing XT-I led to a concentration-dependent shift of enzyme activity towards the cell lysates attended by consistent total intracellular and extracellular XT-I activities. In conclusion, our findings provide a first insight into the XT-I secretion process regulated by a cysteine protease and may contribute to understanding the biological and pathological role of this process.     

Retention of fibroblast growth factor 3 in the Golgi complex may regulate its export from cells.

The fibroblast growth factors (FGFs) fall into two distinct groups with respect to their mode of release from cells. Whereas FGF1 and FGF2 lack conventional signal peptides, the remaining members have typical features of secreted proteins. However, the behavior of mouse FGF3 is anomalous, since, despite entering the secretory pathway and undergoing primary glycosylation, its release from transfected COS-1 cells is very inefficient compared with that of FGF4 and FGF5. To investigate the unusual properties of FGF3, we analyzed the processing, secretion, and intracellular localization of a series of site-directed mutants as well as chimeras produced by fusing parts of FGF3, FGF4, and FGF5. Wild-type FGF3 was shown to accumulate in an immature form in the Golgi complex, from where it is slowly released into the extracellular matrix. Removing or relocating the Asn-linked glycosylation site further impaired its release, and exchanging the signal peptide or carboxy terminus had little effect. In contrast, a chimeric protein with an amino terminus from FGF5 was efficiently secreted and biologically active in cell transformation assays. The data suggest that a structural feature of FGF3 involving the amino-terminal region and glycosylation site has a significant bearing on its passage through the Golgi complex and may regulate the secretion of the ligand.     

The nonclassic secretion of thioredoxin is not sensitive to redox state

Thioredoxin (Trx) is a cytosolic, redox-active protein that is secreted from many cells and has several extracellular functions. In activated lymphocytes, the pathway of secretion does not involve the Golgi apparatus. Levels of extracellular Trx are decreased by the antioxidant N-acetylcysteine. Hence, the secretion of Trx could be altered by the redox status of the cell or the protein. To study Trx mutants, we characterized the secretion of human Trx from Chinese hamster ovary cells. Secretion of human Trx is unaffected by brefeldin A, slow but efficient, and sensitive to low temperature and factors in serum. We demonstrate that N-acetylcysteine reduces the cellular level of Trx but not the proportion secreted; thus this chemical does not block the nonclassic pathway for Trx secretion. Furthermore, we find that mutations in either the active site or the dimerization site of Trx do not alter its secretion. Thus the nonclassic secretion of Trx is not dependent on the redox status of either the cell or the protein.     

Maturation of lipoprotein lipase. Expression of full catalytic activity requires glucose trimming but not translocation to the cis-Golgi compartment.

The relationship between maturation of lipoprotein lipase (LPL) and its translocation from the endoplasmic reticulum (ER) to the Golgi complex was determined by measuring lipolytic activity under conditions preventing transport of the enzyme from the ER to the Golgi compartment. In the presence of brefeldin A, a reagent that inhibits movement of proteins from the ER and causes the disassembly of the Golgi complex, pro-5 Chinese hamster ovary cells accumulated catalytically active LPL, while secretion of the enzyme was effectively blocked. LPL retained intracellularly by brefeldin A treatment possessed oligosaccharide chains that were processed to the complex form by the Golgi enzymes redistributed into the ER. At 16 degrees C, a condition disrupting protein transport to the cis-Golgi, the retained enzyme again remained catalytically active although the oligosaccharides remained in the high mannose form. Lastly, attachment of the specific ER retention signal KDEL (Lys-Asp-Glu-Leu) to the carboxyl terminus of LPL also resulted in intracellularly retained enzyme that was fully active. The importance of oligosaccharide processing for attainment of LPL catalytic activity in vitro was also determined. LPL was active and secreted when trimming of the mannose residues was inhibited by deoxymannojirimycin and when addition of complex sugars was blocked using Chinese hamster ovary mutants (lec1 and lec2), indicating that these processing events are not necessary for the expression of a functional enzyme. However, blocking glucose removal by glucosidase inhibitors (castanospermine and N-methyl-deoxynojirimycin) resulted in a significant reduction in LPL specific activity and secretion. Thus, glucose trimming of LPL oligosaccharides is essential for enzyme activation; however, further oligosaccharide processing or translocation of the enzyme to the cis-Golgi is not required for full expression of lipolytic activity in vitro.     

Intracellular turnover, novel secretion, and mitogenically active intracellular forms of v-sis gene product in simian sarcoma virus-transformed cells. Implications for intracellular loop autocrine transformation

In simian sarcoma virus (SSV)-transformed cells (SSV-NRK, SSV-NIH 3T3, and SSV-NP1 cells), the v-sis gene product was synthesized as a 36-kDa glycopolypeptide with one endoglycosidase (Endo) H-sensitive oligosaccharide chain and formed a dimer (p72) with a half-time of less than 5 min. p72 was proteolytically processed to generate sequentially p68 and p58 in the endoplasmic reticulum/Golgi complex, p44 in the post-Golgi complex compartments, and p27 in an endosomal/lysosomal compartment. A portion (20-30%) of p72 and p68 later became Endo H-resistant but Endo F-sensitive. During processing, the v-sis gene products exhibited rapid turnover, possibly in the endoplasmic reticulum and/or Golgi complex. The rate of turnover correlated with the tumorigenicity previously reported in these SSV-transformed cells. All three SSV-transformed cells secreted v-sis gene product (p44). p44 was secreted but remained tightly associated with the cell surface. This novel secretion provided an efficient system for the interaction of p44 with the cell surface platelet-derived growth factor receptor which resulted in the intracellular formation of p27. A fraction of secreted p44 was converted extracellularly to a 27-kDa product (extracellular p27) after a longer time in culture. The identical N-terminal amino acid sequence of p44 and extracellular p27 (H2N-SLGSLSVAEPAMIA) indicated a preferential site (Lys110-Arg111) for the proteolytic processing. The intracellular turnover of the v-sis gene product and its correlation with tumorigenicity as well as the demonstration of mitogenically active intracellular forms of v-sis gene product support the hypothesis of intracellular loop autocrine transformation.     

Secretion of unhydroxylated chick tendon procollagen.

The secretion of unhydroxylated procollagen at 37° by isolated chick tendon fibroblasts independent of protein synthesis was examined. The data showed that intact molecules were secreted and that their degradation was an extracellular event. The kinetics of secretion indicated that most of the secreted procollagen appeared in the medium during the initial 30 min following inhibition of protein synthesis and only an additional 35% reached the extracellular space in the subsequent 90 min. The pattern of secretion suggested the existence of an intracellular binding site for the unhydroxylated molecules which was saturated during the early period of secretion. It is speculated that such a binding site could be the enzyme prolyl hydroxylase which has a high affinity for unhydroxylated procollagen at 37°.     

Golgi apparatus-localized synaptotagmin 2 is required for unconventional secretion in Arabidopsis

Background

Most secretory proteins contain signal peptides that direct their sorting to the ER and secreted via the conventional ER/Golgi transport pathway, while some signal-peptide-lacking proteins have been shown to export through ER/Golgi independent secretory pathways. Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus that is active against both prokaryotic and eukaryotic cells. The hygromycin phosphotransferase (HYG^R) can phosphorylate and inactivate the hygromycin B, and has been widely used as a positive selective marker in the construction of transgenic plants. However, the localization and trafficking of HYG^R in plant cells remain unknown. Synaptotagmins (SYTs) are involved in controlling vesicle endocytosis and exocytosis as calcium sensors in animal cells, while their functions in plant cells are largely unclear.

Methodology/Principal Findings

We found Arabidopsis synaptotagmin SYT2 was localized on the Golgi apparatus by immunofluorescence and immunogold labeling. Surprisingly, co-expression of SYT2 and HYG^R caused hypersensitivity of the transgenic Arabidopsis plants to hygromycin B. HYG^R, which lacks a signal sequence, was present in the cytoplasm as well as in the extracellular space in HYG^R-GFP transgenic Arabidopsis plants and its secretion is not sensitive to brefeldin A treatment, suggesting it is not secreted via the conventional secretory pathway. Furthermore, we found that HYG^R-GFP was truncated at carboxyl terminus of HYG^R shortly after its synthesis, and the cells deficient SYT2 failed to efficiently truncate HYG^R-GFP,resulting in HYG^R-GFP accumulated in prevacuoles/vacuoles, indicating that SYT2 was involved in HYG^R-GFP trafficking and secretion.

Conclusion/Significance

These findings reveal for the first time that SYT2 is localized on the Golgi apparatus and regulates HYG^R-GFP secretion via the unconventional protein transport from the cytosol to the extracelluar matrix in plant cells.     

Secretory function of autophagy in innate immune cells

Eukaryotic cells utilize two main secretory pathways to transport proteins to the extracellular space. Proteins with a leader signal sequence often undergo co‐translational transport into the endoplasmic reticulum (ER), and then to the Golgi apparatus before they reach their destination. This pathway is called the conventional secretory pathway. Proteins without signal peptides can bypass this ER‐Golgi system and are secreted by a variety of mechanisms collectively called the unconventional secretory pathway. The molecular mechanisms of unconventional secretion are emerging. Autophagy is a conserved bulk degradation mechanism that regulates many intracellular functions. Recent evidence implicates autophagy in the secretory pathway. This review focuses on potential secretory roles of autophagy and how they could modulate the functions of innate immune cells that secrete a wide range of mediators in response to environmental and biological stimuli. We provide a brief overview of the secretory pathways, enumerate the potential mechanistic themes by which autophagy interacts with these pathways and describe their relevance in the context of innate immune cell function.     

Intracellular lipidation of newly synthesized apolipoprotein A-I in primary murine hepatocytes

Hepatocytes, which are the main site of apolipoprotein (apo)A-I and ATP-binding cassette transporter A1 (ABCA1) expression, are also the main source of circulating high density lipoprotein. Here we have characterized the intracellular lipidation of newly synthesized apoA-I, in primary hepatocytes cultured with [3H]choline to label choline-phospholipids, low density lipoprotein-[3H]cholesterol to label the cell surface, or [3H]mevalonate to label de novo synthesized cholesterol. Phospholipidation of apoA-I is significant and most evident in endoplasmic reticulum (ER) and medial Golgi, both in the lumen and on the membrane fractions of the ER and medial Golgi. In the presence of cycloheximide, endogenous apoA-I is substantially phospholipidated intracellularly but acquires some additional lipid after export out of the cell. In cells labeled with low density lipoprotein-[3H]cholesterol, intracellular cholesterol lipidation of apoA-I is entirely absent, but the secreted apoA-I rapidly accumulates cholesterol after secretion from the cell in the media. On the other hand, de novo synthesized cholesterol can lipidate apoA-I intracellularly. We also showed the interaction between apoA-I and ABCA1 in ER and Golgi fractions. In hepatocytes lacking ABCA1, lipidation by low density lipoprotein-cholesterol was significantly reduced at the plasma membrane, phospholipidation and lipidation by de novo synthesized sterols were both reduced in Golgi compartments, whereas ER lipidation remained mostly unchanged. Therefore, the early lipidation in ER is ABCA1 independent, but in contrast, the lipidation of apoA-I in Golgi and at the plasma membrane requires ABCA1. Thus, we demonstrated that apoA-I phospholipidation starts early in the ER and is partially dependent on ABCA1, with the bulk of lipidation by phospholipids and cholesterol occurring in the Golgi and at the plasma membrane, respectively. Finally, we showed that the previously reported association of newly synthesized apoA-I and apoB (Zheng, H., Kiss, R. S., Franklin, V., Wang, M. D., Haidar, B., and Marcel, Y. L. (2005) J. Biol. Chem. 280, 21612-21621) occurs after secretion at the cell surface.     

The rate of bulk flow from the endoplasmic reticulum to the cell surface

Tripeptides containing the acceptor sequence for Asn-linked glycosylation (Asn-X-Ser/Thr) were added to CHO and HepG2 cells. The tripeptides were glycosylated in the ER and then secreted into the medium, via the Golgi complex in which the oligosaccharide chains were processed. The half-time for secretion, approximately 10 min, was faster than that of known proteins transported through the same pathway. Since much evidence suggests that oligosaccharide chains are not signals for transport, it appears that no signal is necessary for rapid and efficient transport from the ER to the Golgi, or from the Golgi to the cell surface. Rather, it appears that proteins retained as permanent residents en route through the ER-Golgi transport pathway must contain specific retention signals.