A single amino acid substitution in the variable region of the light chain specifically blocks immunoglobulin secretion.

Although immunoglobulin light chains are usually secreted in association with heavy chains, free light chains can be secreted by lymphocytes. To identify the structural features of light chains that are essential for their secretion, we mutated a conserved sequence in the variable domain of a lambda I light chain. The effects of the mutations on secretion were assayed by transient expression in COS-1 cells. One mutant (AV60), which replaced Ala-60 with Val, was secreted as efficiently as wild-type lambda I by transfected COS-1 cells. This result was not surprising because secreted lambda II chains contain valine in this position. However, a second lambda I mutant (AV60FS62), which replaced Phe-62 with Ser as well as Ala-60 with Val, was not secreted. This mutant was arrested in the endoplasmic reticulum, as judged by immunofluorescence and by its association with a lumenal endoplasmic reticulum protein, immunoglobulin heavy chain binding protein (BiP). The defect in secretion was not due to gross misfolding of the lambda I chain, since cells cotransfected with AV60FS62 and an immunoglobulin heavy chain gene produced functional antigen-binding antibodies. These assembled IgM molecules were still not secreted. Hence, the replacement of Phe-62 with Ser specifically affects a determinant on the lambda I light chain that is necessary for the intracellular transport of this molecule.     

Human macrophage colony-stimulating factor is expressed at and shed from the cell surface

Surface membrane-associated growth factors are being recognized as important for developmental processes, including cell assembly, differentiation, and growth. To investigate the role of membrane-bound macrophage colony-stimulating factor (M-CSF) in myelopoiesis, and whether this factor is released from the cell surface in association with shed membrane-derived vesicles, COS-1 cells were transfected with cDNAs for M-CSF-tau (containing the transmembrane domain) or a soluble mutant form of the molecule lacking the transmembrane domain ([s]M-CSF- alpha). COS-1 cells transfected with either cDNA released activity into the spent culture medium. Conditioned medium was separated by centrifugation into supernatants and pellets were found to contain plasma membrane-derived vesicles by transmission electron microscopy. When medium fractions were assayed in marrow cultures, activity was localized to shed plasma membrane-derived vesicles in medium conditioned by cells transfected with cDNA for M-CSF-tau and in the vesicle-free supernatants of medium conditioned by cells transfected with cDNA for [s]M-CSF-alpha. In addition, nuclear, mitochondrial, and plasma membrane subfractions of stably transfected cells were prepared and assayed for activity. Concentration-dependent stimulation of macrophage colony formation was observed with purified plasma membranes (but not nuclear or cytosolic proteins) from cells transfected with cDNA for M-CSF-tau. By contrast, membranes from untransfected cells and cells transfected with cDNA for [s]M-CSF-alpha or control DNA expressed no activity. Together, the data indicate that human M-CSF is expressed at the cell surface and exfoliated in association with surface membrane- derived vesicles.     

Heterologous in vivo processing of human preproendothelin 1 into bioactive peptides.

Endothelin (ET) is an extremely potent vasoconstrictor peptide of 21 amino acids, originally found in the supernatant of cultured vascular endothelial cells. To gain insights into its biosynthetic pathway, we expressed a synthetic RNA coding for the 212-amino acid precursor of human ET-1 (preproET-1) in Xenopus oocytes. Cell homogenates and oocyte incubation medium were tested by RIA using an anti-ET-1 serum. ET-1-like immunoreactivity was detected in oocytes injected with preproET-1 synthetic RNA but not in control oocytes and was much higher in medium than in cell homogenates. When preproET-1 was expressed in oocytes treated with monensin, a dramatic decrease in secretion of immunoreactive material was observed, indicating that secretion is mediated by the Golgi complex. ET-1-like immunoreactive material present in oocyte incubation medium was fractionated by reverse-phase HPLC into two main peaks, corresponding to the retention times of human big ET-1 and ET-1. Incubation medium of oocytes expressing the synthetic preproET-1 RNA elicited a characteristic vasoconstrictor response on rabbit vena cava, consistent with the biological activity that would be predicted from the amount of ET-1-like immunoreactivity measured. These results suggest that common pathways of ET maturation exist in widely different cells and that Xenopus oocytes may represent a useful tool in studying the cell biology of ET-1 synthesis.     

Proteolytic processing of big endothelin-3 by the kell blood group protein.

Kell blood group protein shares a consensus sequence (H.E.X.X.H) with a large family of zinc-dependent endopeptidases. Kell has closest homology with neutral endopeptidase 24.11, endothelin converting enzyme-1 (ECE-1), and the PEX gene product that, as a group, comprise the M13 subfamily of mammalian neutral endopeptidases. The proteolytic activity of the M13 members, but not of Kell, has been previously demonstrated. A secreted form of wild-type Kell protein (s-Kell), devoid of the intracellular and transmembrane domains, was expressed in sf9 cells. As a negative control, an inactive mutant Kell protein (E582G) was expressed. As determined by N-terminal amino acid sequencing and mass spectrometry of the cleaved products, wild-type s-Kell, but not the control mutant protein, specifically cleaved big endothelin-3 (ET-3) at Trp(21)-Ile(22), yielding ET-3, and, to a much lesser extent, also cleaved big ET-1 and big ET-2 at Trp(21)-Val(22), yielding ET-1 and ET-2. Enzymatic activity was partially inhibited by phosphoramidon. s-Kell has an acidic pH optimum (pH 6.0 to 6.5). Like the recombinant protein, red blood cells of common Kell phenotype also preferentially process big ET-3, in contrast to Ko (null) cells that do not. These data demonstrate that the Kell blood group protein is a proteolytic enzyme that processes big ET-3, generating ET-3, a potent bioactive peptide with multiple biological roles.     

In vivo expression of mutant preproendothelins: hierarchy of processing events but no strict requirement of Trp-Val at the processing site.

Endothelin-1 (ET-1), a 21-residue vasoconstrictor peptide, originates in human cells from a 212-amino acid precursor (preproET-1). Big ET-1, an intermediate form of 38 amino acids, is generated by cleavage at basic-pair residues of proET-1, while a specific "ET-converting enzyme" was proposed to process the unusual Trp-Val site at positions 21 and 22 of big ET-1. We have previously shown that expression of synthetic RNA encoding human preproET-1 in Xenopus oocytes results in secretion of putative ET-1 and big ET-1. Here, to further dissect the processing pathway of preproET-1, we designed and expressed in oocytes a set of preproET-1 mutants. Four mutants affecting the Trp-Val site always originated putative ET-1(s) at levels comparable to the wild type, suggesting that there is only a conformational requirement for cleavage at this site. An Arg-->Ile mutation at the basic-pair site after the C terminus of big ET-1 fully inhibited the formation of both big ET-1 and ET-1, indicating that processing at this site is an early event and that big ET-1 is an obligate intermediate for the synthesis of ET-1 in vivo. Also, a truncated mutant bearing a stop codon after the C terminus of the big ET-1 sequence was totally stable and further processed into mature big ET-1 and ET-1, indicating that the second part of the precursor is not necessary for maturation.     

Small GTP-binding proteins in human endothelial cells

Small GTP-binding proteins of the Ras superfamily control an extensive number of intracellular events by alternating between GDP- and GTP-bound conformation. The presence of members of this protein family was examined in human umbilical vein endothelial cells employing RT-PCR. Sequence analysis of 215 cDNA clones revealed the presence of a total of 28 different partial cDNAs encoding small GTP-binding proteins. Two sequences corresponded to novel isoforms of Rab2 and Rab9. In addition, human analogues of Rab4b, Rab7, Rab9, Rab14 and Rab15 were identified. Besides Rab proteins, members of other subfamilies were detected as well. As a first step towards elucidation of the function of the different small GTP-binding proteins identified we have isolated full length cDNA corresponding to Rab30 from a human endothelial cell cDNA library. In order to assess the subcellular localization of Rab30, we expressed epitope-tagged Rab30 cDNA in monkey kidney COS-1 cells. Immunoelectron-microscopy of transfected COS-1 cells indicated that Rab30 is associated with Golgi stacks.     

Phosphoramidon blocks the pressor activity of porcine big endothelin-1-(1-39) in vivo and conversion of big endothelin-1-(1-39) to endothelin-1-(1-21) in vitro.

In porcine aortic endothelial cells, the 21-amino acid peptide endothelin-1 (ET-1) is formed from a 39-amino acid intermediate called "big endothelin-1" (big ET-1) by a putative ET-converting enzyme (ECE) that cleaves the 39-mer at the bond between Trp-21 and Val-22. Since big ET-1 has only 1/100-1/150th the contractile activity of ET-1, inhibition of ECE should effectively block the biological effects of ET-1. Big ET-1 injected intravenously into anesthetized rats produces a sustained pressor response that presumably is due to conversion of big ET-1 into ET-1 by ECE. We determined the type of protease activity responsible for this conversion by evaluating the effectiveness of protease inhibitors in blocking the pressor response to big ET-1 in ganglion-blocked anesthetized rats. The serine protease inhibitor leupeptin, the cysteinyl protease inhibitor E-64, and the metalloprotease inhibitors captopril and kelatorphan were all ineffective at blocking the pressor response to big ET-1. However, the metalloprotease inhibitors phosphoramidon and thiorphan dose-dependently inhibited the pressor response to big ET-1, although phosphoramidon was substantially more potent than thiorphan. None of the inhibitors blocked the pressor response to ET-1 and none had any effect on mean arterial pressure when administered alone. In a rabbit lung membrane preparation, ECE activity was identified that was blocked by the metalloprotease inhibitors phosphoramidon and 1,10-phenanthroline in a concentration-dependent manner. This enzyme converted big ET-1 to a species of ET that comigrated on HPLC with ET-1 and produced an ET-like contraction in isolated rat aortic rings. Our results suggest that the physiologically relevant ECE is a metalloprotease.     

Identification of endothelin 1 and big endothelin 1 in secretory vesicles isolated from bovine aortic endothelial cells.

Vesicles containing endothelin 1 (ET-1) were isolated from bovine aortic endothelial cells (BAECs) by fractionation of homogenates on sucrose density gradients by ultracentrifugation. The vesicles were localized at the 1.0/1.2 M sucrose interface using a specific anti-ET-1-(16-21) RIA. Identification of ET-1 and big ET-1 in this fraction was confirmed by HPLC analysis combined with RIA. Morphological examination of the ET-1-enriched fraction by electron microscopy identified clusters of vesicles approximately 100 nm in diameter. Immunostaining of ultrathin cryosections prepared from the vesicle fraction for ET-1 or big ET-1 showed clusters of 15-nm gold particles attached to or within vesicles. Immunofluorescence staining of whole BAECs using a specific ET-1-(16-21) IgG purified by affinity chromatography revealed punctate granulation of the cell cytoplasm viewed under light microscopy. This distinct pattern of staining was shown by confocal light microscopy to be intracellular. Immunofluorescence staining of whole cells with a polyclonal antiserum for big ET-1-(22-39) showed a defined perinuclear localization of precursor molecule. Hence, several different approaches have demonstrated that ET-1 and big ET-1 are localized within intracellular vesicles in BAECs, suggesting that these subcellular compartments are an important site for processing of big ET-1 by endothelin-converting enzyme.     

Isolation of a cDNA encoding a murine UDPgalactose:beta-D-galactosyl- 1,4-N-acetyl-D-glucosaminide alpha-1,3-galactosyltransferase: expression cloning by gene transfer.

We have developed a genetic approach to isolate cloned cDNA sequences that determine expression of cell surface oligosaccharide structures and their cognate glycosyltransferases. A cDNA library was constructed in a mammalian expression vector by using mRNA from a murine cell line known to express a UDPgalactose:beta-D-galactosyl-1,4-N-acetyl-D-glucosaminide alpha-1,3-galactosyltransferase [(alpha 1-3)GT; EC 2.4.1.151]. This library was transfected into COS-1 cells, which lack expression of (alpha 1-3)GT. Transfected cells containing functional (alpha 1-3)GT cDNAs were detected and isolated with a lectin that recognizes the surface-expressed glycoconjugate product of the (alpha 1-3)GT enzyme. One cloned (alpha 1-3)GT cDNA was rescued from lectin-positive transfected cells. This cDNA contains a single long open reading frame that predicts a 394-amino-acid protein. No significant primary structure similarities were identified between this protein and other known sequences. However, the protein predicts a type II transmembrane topology similar to two other mammalian glycosyltransferases. This topology places the large COOH-terminal domain within the Golgi lumen; this domain was shown to be catalytically active when expressed in COS-1 cells as a portion of a secreted protein A fusion peptide. Biochemical analysis confirmed that this enzyme catalyzes a transglycosylation reaction between UDP-Gal and Gal(beta 1-4)GlcNAc to form Gal(alpha 1-3)Gal(beta 1-4)GlcNAc. This cloning approach may be generally applicable to the isolation of cDNAs encoding other mammalian glycosyltransferases.     

A major C-peptide deletion prevents secretion of a mutant human proinsulin from transfected monkey kidney cells

The biosynthesis and secretion of human proinsulin and a mutant human proinsulin with a major deletion in the C-peptide, (des 38-62)proinsulin, was studied in monkey kidney cells (Cos-7) transfected with cDNAs encoding the respective normal or mutant human preproinsulins. Transfected cells were labelled with [3H]leucine, and insulin-like material was immunoprecipitated and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. It was found that the prepeptide was removed from both the normal and mutant preproinsulins, and that there was no further processing to insulin. The normal proinsulin was rapidly released from the transfected cells, with little intracellular accumulation, while the mutant proinsulin was retained within the cell, with only small quantities of radio-labelled material in the medium. The intracellular mutant proinsulin was membrane bound and located predominantly within a microsomal fraction. These results suggest that C-peptide plays an important role in the efficient transfer of proinsulin through the early stages of the secretory pathway.     

Isolation and characterization of a human interleukin cDNA clone, homologous to mouse B-cell stimulatory factor 1, that expresses B-cell- and T-cell-stimulating activities.

A cDNA sequence coding for a human interleukin has been isolated from a concanavalin A-activated human T-cell cDNA library based on homology with a mouse interleukin cDNA that expresses B-cell stimulatory factor 1 (BSF-1) activity and T-cell- and mast-cell-stimulating activities. The human cDNA contains a single open reading frame encoding a protein of 153 amino acid residues including a putative signal peptide. Amino acid sequences of the mouse and human polypeptides, deduced from their cDNAs, share extensive homology with the exception of about 40 amino acid residues near the middle portion, which share little homology. Supernatant of COS-7 monkey cells transfected with the human cDNA clone stimulated proliferation of human helper T-cell clones and of anti-IgM-activated human B cells, two properties of mouse BSF-1 on mouse cells. These results indicate that this human cDNA clone encodes a protein structurally and functionally homologous to mouse BSF-1.     

Inhibition of growth hormone (GH) secretion by a mutant GH-I gene product in neuroendocrine cells containing secretory granules: an implication for isolated GH deficiency inherited in an autosomal dominant manner.

Isolated GH deficiency (IGHD) type II is a disease inherited in an autosomal dominant manner. Although point mutations at the donor splice site of intron 3 of the GH-I gene have been identified in patients, the mechanism of how such mutations result in severe GH deficiency is unclear. Recently, we identified two mutations in Japanese patients with IGHD type II, G to A substitutions at the first (mutA) and fifth (mutE) nucleotides of intron 3. Messenger ribonucleic acids skipping exon 3 were transcribed from both mutant GH-I genes. We studied in this report the synthesis and secretion of GH encoded by the mutant GH-I genes and tested whether inhibition of wild-type GH secretion by mutant products could be demonstrated in cultured cell lines. A metabolic labeling study in COS-1 cells revealed that a mutant GH with a reduced molecular mass was synthesized from the mutant messenger ribonucleic acid and retained in the cells for at least 6 h. On the other hand, the wild-type GH was rapidly secreted into the medium. Coexpression of mutant and wild-type GH did not result in any inhibition of wild-type GH secretion in COS-1 or HepG2 cells. However, coexpression of mutant GH resulted in significant inhibition of wild-type GH secretion in somatotroph-derived MtT/S cells as well as in adrenocorticotroph-derived AtT-20 cells, without affecting cell viability. We conclude that the dominant negative effect of mutant GH on the secretion of wild-type GH is at least in part responsible for the pathogenesis of IGHD type II. Our results also suggest that neuroendocrine cell type-specific mechanisms, including intracellular storage of the secretory proteins, are involved in the inhibition.     

Subunit structure of the erythropoietin receptor analyzed by 125I-Epo cross-linking in cells expressing wild-type or mutant receptors

To analyze the structure of the murine erythropoietin receptor (EpoR), wild-type or mutant EpoR cDNAs were expressed in cell lines, and the proteins that cross-linked with 125I-labeled erythropoietin (Epo) were analyzed by immunoprecipitation using an antibody against the intracellular region of the cloned EpoR. COS-7 cell transfectants expressing the wild-type EpoR showed two major cross-linked species of 145 and 110 Kd, both of which were recognized by the antibody against the cloned EpoR after denaturation under reducing conditions. Furthermore, a reduction in sizes of both cross-linked bands was observed in COS-7 transfectants expressing a mutant receptor with an internal deletion, thus indicating that both species contain the cloned EpoR. COS-7 cells expressing mutant receptors with carboxy-terminal deletions showed cross-linked bands corresponding to the smaller species of the two observed in cells expressing the wild-type receptor. In contrast to COS-7 cell transfectants, DA3 cells expressing wild-type or mutant EpoR cDNAs showed an additional cross-like species of 130 Kd. The size of this species was not altered by deletions in EpoR, showing that it did not contain EpoR. The 130-Kd cross-linked band, which would contain a 95-Kd protein, was also observed in a murine erythroleukemia cell line, D1B. These results suggest that Epo associates with a second component of 95 Kd, which is specifically expressed in hematopoietic cells.     

Homology modeling and functional testing of an ABCA1 mutation causing Tangier disease

Objective

To investigate the impact of the p.R1068H mutation on the structure and function of the ATP-binding cassette A1 (ABCA1) protein.

Methods

A homology model of the nucleotide binding domains of ABCA1 was constructed to identify the three-dimensional orientation of R1068. Cholesterol efflux assays were performed on fibroblasts obtained from members of a Tangier disease (TD) family carrying the p.R1068H mutation and in HEK293 cells transfected with a p.R1068H mutant cDNA vector. Confocal microscopy was used to investigate the localisation of the wildtype and mutant p.R1068H protein in HEK293 cells.

Results

Sequence alignments and modeling indicated residue R1068 to be located in an α-helix downstream of the Walker B motif in the first nucleotide binding domain (NBD-1), in a position to form ionic interactions with D1092 and E1093. Cholesterol efflux studies showed the efflux from TD fibroblasts and HEK293 cells expressing the mutant p.R1068H protein to be markedly reduced compared to wildtype. Localisation of the mutant p.R1068H protein in HEK293 cells showed intracellular retention of the protein indicating a defect in trafficking to the plasma membrane.

Conclusion

Homology modeling of the ABCA1 protein showed that the p.R1068H mutation would likely disrupt the conformation of NBD-1. Functional studies of p.R1068H showed a lack of cholesterol efflux function due to defective trafficking to the plasma membrane, most likely caused by impaired oligomerisation.     

Granulocyte-macrophage colony-stimulating factor mRNA stabilization enhances transgenic expression in normal cells and tissues

To increase transgenic production of granulocyte-macrophage colony- stimulating factor (GM-CSF), we mutated the mRNA's 3'-untranslated region, AUUUA instability elements. Expression vectors containing human or murine GM-CSF cDNAs coding for wild-type (GM-AUUUA) or mutant versions with reiterated AUGUA repeats (GM-AUGUA) were transfected into cells in culture or animals using particle-mediated gene-transfer technology. Normal peripheral blood mononuclear cells accumulated 20- fold greater levels of GM-CSF mRNA and secreted comparably greater amounts of cytokine after transfection with hGM-AUGUA expression vectors versus hGM-AUUUA. hGM-AUGUA mRNA was fivefold more stable (t 1/2 = 95 minutes) than hGM-AUUUA mRNA (t 1/2 = 20 minutes), accounting for elevated steady-state levels. Transfection site extracts and serum samples obtained 24 hours after gene transfer of hGM-AUGUA cDNA into mouse skin contained greater than 32 ng/mL and 650 pg/mL of GM-CSF protein, respectively, compared with 0.33 ng/mL and less than 8 pg/mL for hGM-AUUUA cDNA. GM-CSF produced from mGM-AUGUA cDNA transfected into rat abdominal epidermis induced a profound neutrophil infiltrate. These data suggest a novel strategy for enhanced production of biologically active cytokines by normal cells after in vivo gene transfer.     

J-HNP-1多肽的重组及体外抗菌作用研究

目的:将α-防御素-1(HNP-1)基因与J链基因进行重组使其成为一种新的杀菌肽分子J-HNP-1,并将其构建于能有效表达和分泌活性J-HNP-1杀菌肽的哺乳动物细胞表达系统．方法:应用PCR技术从不同的质粒中获得J链基因和 HNP-1基因,然后应用重组PCR技术将这两个不同的基因连接在一起而成为J-HNP-1基因．将此基因克隆入哺乳动物细胞表达载体pcDNA3．1(-)／Myc-HisC中．用脂质体转染法将此重组子导入COS-7细胞,分别从 mRNA和蛋白质水平采用RT-PCR和Western blot分析 J-HNP-1的表达情况,并体外检测细胞可溶性蛋白及培养上清的抗菌活性．结果:获得的J链基因和HNP-1基因大小分别为489和 297 bp．采用RT-PCR法从被转染的细胞中扩增出一条约786 bp的片段,其大小与预测相符;采用Western blot 印迹法．用抗His抗体检测到细胞可溶性蛋白及培养上清在约Mr 24 000处有强反应条带显示;抗菌活性检测显示,经rpcDNA3．1(-)／Myc-HisC／J-HNP-1转染的 COS-7细胞的可溶性蛋白和培养上清均有明显的抗菌活性,抑菌圈的直径分别为34和43 mm．结论:重组J-HNP-1基因构建到哺乳动物细胞表达系统,并体外表达,具有抑菌作用．     

Evidence for intracellular endothelin-converting enzyme-2 expression in cultured human vascular endothelial cells

We have previously reported the intracellular localization of the endothelin-converting enzyme-1 (ECE-1) in human umbilical vein endothelial cells. In the present study, we provide the first immunocytochemical and biochemical evidence for the presence of ECE-2 in human cells. ECE activity was determined by conversion of exogenously added big endothelin-1 (big ET-1) to ET-1 in subcellular fractions obtained by sucrose density gradient centrifugation of human umbilical vein endothelial cell homogenates. ECE-1 and ECE-2 can be differentiated by pH dependence for optimal activity and by sensitivity to phosphoramidon, which shows selectivity for ECE-2 over ECE-1 and PD159790, a novel ECE-1 selective inhibitor. Optimal ECE activity was measured at pH 6.0, a value intermediate between that reported for ECE-1 (pH 6.8) and ECE-2 (pH 5.5), indicating expression of both enzymes. At pH 6.9, conversion of big ET-1 was inhibited markedly by 30 micromol/L PD159790 and by 100 micromol/L phosphoramidon but not by 0.1 micromol/L phosphoramidon. In contrast, ECE activity was unaffected by 30 micromol/L PD159790 but was inhibited markedly by 0.1 and 100 micromol/L phosphoramidon at pH 5. 4 (IC50 1.5 nmol/L), consistent with ECE-2 activity. Confocal microscopy revealed a punctate pattern of ECE-2-like immunoreactive staining in the cell cytosol, suggesting localization to secretory vesicles with a possible role in processing big ET-1 while in transit to the cell surface via the constitutive secretory pathway.     

Structure and function in rhodopsin: kinetic studies of retinal binding to purified opsin mutants in defined phospholipid-detergent mixtures serve as probes of the retinal binding pocket

In the current standard procedure for preparation of mammalian rhodopsin mutants, transfected COS-1 cells expressing the mutant opsin genes are treated with 5 microM 11-cis-retinal before detergent solubilization for purification. We found that binding of 11-cis-retinal to opsin mutants with single amino acid changes at Trp-265 (W265F,Y,A) and a retinitis pigmentosa mutant (A164V) was far from complete and required much higher concentrations of 11-cis-retinal. By isolation of the expressed opsins in a stable form, kinetic studies of retinal binding to the opsins in vitro have been carried out by using defined phospholipid-detergent mixtures. The results show wide variation in the rates of 11-cis-retinal binding. Thus, the in vitro reconstitution procedure serves as a probe of the retinal-binding pocket in the opsins. Further, a method is described for purification and characterization of the rhodopsin mutants after retinal binding to the opsins in vitro.     

Human disease-causing NOG missense mutations: effects on noggin secretion, dimer formation, and bone morphogenetic protein binding

Secreted noggin protein regulates bone morphogenetic protein activity during development. In mice, a complete loss of noggin protein leads to multiple malformations including joint fusion, whereas mice heterozygous for Nog loss-of-function mutations are normal. In humans, heterozygous NOG missense mutations have been found in patients with two autosomal dominant disorders of joint development, multiple synostosis syndrome (SYNS1) and a milder disorder proximal symphalangism (SYM1). This study investigated the effect of one SYNS1 and two SYM1 disease-causing missense mutations on the structure and function of noggin. The SYNS1 mutation abolished, and the SYM1 mutations reduced, the secretion of functional noggin dimers in transiently transfected COS-7 cells. Coexpression of mutant noggin with wild-type noggin, to resemble the heterozygous state, did not interfere with wild-type noggin secretion. These data indicate that the human disease-causing mutations are hypomorphic alleles that reduce secretion of functional dimeric noggin. Therefore, we conclude that noggin has both species-specific and joint-specific dosage-dependent roles during joint formation. Surprisingly, in contrast to the COS-7 cell studies, the SYNS1 mutant was able to form dimers in Xenopus laevis oocytes. This finding indicates that there also exist species-specific differences in the ability to process mutant noggin polypeptides.