Latent-transforming growth factor beta-binding protein-2 (LTBP-2) is required for longevity but not for development of zonular fibers.

Latent-transforming growth factor beta-binding protein 2 (LTBP-2) is a major component of arterial and lung tissue and of the ciliary zonule, the system of extracellular fibers that centers and suspends the lens in the eye. LTBP-2 has been implicated previously in the development of extracellular microfibrils, although its exact role remains unclear. Here, we analyzed the three-dimensional structure of the ciliary zonule in wild type mice and used a knockout model to test the contribution of LTBP-2 to zonule structure and mechanical properties. In wild types, zonular fibers had diameters of 0.5-1.0 micrometers, with an outer layer of fibrillin-1-rich microfibrils and a core of fibrillin-2-rich microfibrils. LTBP-2 was present in both layers. The absence of LTBP-2 did not affect the number of fibers, their diameters, nor their coaxial organization. However, by two months of age, LTBP-2-depleted fibers began to rupture, and by six months, a fully penetrant ectopia lentis phenotype was present, as confirmed by in vivo imaging. To determine whether the seemingly normal fibers of young mice were compromised mechanically, we compared zonule stress/strain relationships of wild type and LTBP-2-deficient mice and developed a quasi-linear viscoelastic engineering model to analyze the resulting data. In the absence of LTBP-2, the ultimate tensile strength of the zonule was reduced by about 50%, and the viscoelastic behavior of the fibers was altered significantly. We developed a harmonic oscillator model to calculate the forces generated during saccadic eye movement. Model simulations suggested that mutant fibers are prone to failure during rapid rotation of the eyeball. Together, these data indicate that LTBP-2 is necessary for the strength and longevity of zonular fibers, but not necessarily for their formation.

Macrophage secretion of miR-106b-5p causes renin-dependent hypertension.

Myeloid cells are known mediators of hypertension, but their role in initiating renin-induced hypertension has not been studied. Vitamin D deficiency causes pro-inflammatory macrophage infiltration in metabolic tissues and is linked to renin-mediated hypertension. We tested the hypothesis that impaired vitamin D signaling in macrophages causes hypertension using conditional knockout of the myeloid vitamin D receptor in mice (KODMAC). These mice develop renin-dependent hypertension due to macrophage infiltration of the vasculature and direct activation of renal juxtaglomerular (JG) cell renin production. Induction of endoplasmic reticulum stress in knockout macrophages increases miR-106b-5p secretion, which stimulates JG cell renin production via repression of transcription factors E2f1 and Pde3b. Moreover, in wild-type recipient mice of KODMAC/miR106b-/- bone marrow, knockout of miR-106b-5p prevents the hypertension and JG cell renin production induced by KODMAC macrophages, suggesting myeloid-specific, miR-106b-5p-dependent effects. These findings confirm macrophage miR-106b-5p secretion from impaired vitamin D receptor signaling causes inflammation-induced hypertension.

Contribution of metabolic disease to bone fragility in MAGP1-deficient mice.

Microfibril-associated glycoprotein-1 (MAGP1) is an extracellular matrix protein that interacts with fibrillin and is involved in regulating the bioavailability of signaling molecules such as TGFß. Mice with germline MAGP1 deficiency (Mfap2-/-) develop increased adiposity, hyperglycemia, insulin resistance, bone marrow adipose tissue expansion, reduced cancellous bone mass, cortical bone thinning and bone fragility. The goal of this study was to assess whether the Mfap2-/- bone phenotypes were due to loss of MAGP1 locally or secondary to a change in whole body physiology (metabolic dysfunction). To do this, mice with conditional deletion of MAGP1 in the limb skeleton were generated by crossing MAGP1-flox mice (Mfap2lox/lox) with Prx1-Cre mice. Mfap2Prx-/- mice did not show any changes in peripheral adiposity, hyperglycemia or insulin sensitivity, but did have increased bone length and cancellous bone loss that was comparable to the germline Mfap2-/- knockout. Unlike the germline knockout, marrow adiposity, cortical bone thickness and bone strength in Mfap2Prx-/- mice were normal. These findings implicate systemic metabolic dysfunction in the development of bone fragility in germline Mfap2-/- mice. An unexpected finding of this study was the detection of MAGP1 protein in the Mfap2Prx-/- hematopoietic bone marrow, despite the absence of MAGP1 protein in osseous bone matrix and absent Mfap2 transcript expression at both sites. This suggests MAGP1 from a secondary site may accumulate in the bone marrow, but not be incorporated into the bone matrix, during times of regional MAGP1 depletion.

Loss of Elastic Fiber Integrity Compromises Common Carotid Artery Function: Implications for Vascular Aging.

Competent elastic fibers endow central arteries with the compliance and resilience that are fundamental to their primary mechanical function in vertebrates. That is, by enabling elastic energy to be stored in the arterial wall during systole and then to be used to work on the blood during diastole, elastic fibers decrease ventricular workload and augment blood flow in pulsatile systems. Indeed, because elastic fibers are formed during development and stretched during somatic growth, their continual tendency to recoil contributes to the undulation of the stiffer collagen fibers, which facilitates further the overall compliance of the wall under physiologic pressures while allowing the collagen to limit over-distension during acute increases in blood pressure. In this paper, we use consistent methods of measurement and quantification to compare the biaxial material stiffness, structural stiffness, and energy storage capacity of murine common carotid arteries having graded degrees of elastic fiber integrity - normal, elastin-deficient, fibrillin-1 deficient, fibulin-5 null, and elastase-treated. The finding that the intrinsic material stiffness tends to be maintained nearly constant suggests that intramural cells seek to maintain a favorable micromechanical environment in which to function. Nevertheless, a loss of elastic energy storage capability due to the loss of elastic fiber integrity severely compromises the primary function of these central arteries.

Matrix modulation of compensatory lung regrowth and progenitor cell proliferation in mice.

Mechanical stress is an important modulator of lung morphogenesis, postnatal lung development, and compensatory lung regrowth. The effect of mechanical stress on stem or progenitor cells is unclear. We examined whether proliferative responses of epithelial progenitor cells, including dually immunoreactive (CCSP and proSP-C) progenitor cells (CCSP+/SP-C+) and type II alveolar epithelial cells (ATII), are affected by physical factors found in the lung of emphysematics, including loss of elastic recoil, reduced elastin content, and alveolar destruction. Mice underwent single lung pneumonectomy (PNY) to modulate transpulmonary pressure (mechanical stress) and to stimulate lung regeneration. Control mice underwent sham thoracotomy. Plombage of different levels was employed to partially or completely abolish this mechanical stress. Responses to graded changes in transpulmonary pressure were assessed in elastin-insufficient mice (elastin +/-, ELN+/-) and elastase-treated mice with elastase-induced emphysema. Physiological regrowth, morphometry (linear mean intercept; Lmi), and the proliferative responses of CCSP+/SP-C+, Clara cells, and ATII were evaluated. Plombage following PNY significantly reduced transpulmonary pressure, regrowth, and CCSP+/SP-C+, Clara cell, and ATII proliferation following PNY. In the ELN+/- group, CCSP+/SP-C+ and ATII proliferation responses were completely abolished, although compensatory lung regrowth was not significantly altered. In contrast, in elastase-injured mice, compensatory lung regrowth was significantly reduced, and ATII but not CCSP+/SP-C+ proliferation responses were impaired. Elastase injury also reduced the baseline abundance of CCSP+/SP-C+, and CCSP+/SP-C+ were found to be displaced from the bronchioalveolar duct junction. These data suggest that qualities of the extracellular matrix including elastin content, mechanical stress, and alveolar integrity strongly influence the regenerative capacity of the lung, and the patterns of cell proliferation in the lungs of adult mice.

Lysyl oxidase like-1 dysregulation and its contribution to direct inguinal hernia.

OBJECTIVE: The aetiology of inguinal hernia involves changes in collagen turnover and metalloproteinase expression; yet it is not known whether the elastic fibre system could also be affected. This study was designed to compare the expression of tropoelastin (TE), lysyl oxidase-like 1 (LOXL-1) and elastase in the transversalis fascia of patients with and without inguinal hernia. MATERIAL AND METHODS: Transversalis fascia (TF) specimens were obtained from patients undergoing surgery for direct or indirect inguinal hernia (n = 20 each) and from multi-organ donors during organ procurement (controls, n = 16). The specimens were divided according to age (20-40/41-60 years). Tissues were immunohistochemically labelled using anti-tropoelastin, anti-LOXL-1 and anti-elastase antibodies and subjected to Western blot analysis. Relative amounts of LOXL-1 and TE mRNA were determined by real time RT-PCR in cultured cells obtained from the TF of patients and controls. RESULTS: Significantly lower TE and LOXL-1 levels were observed in patients with direct inguinal hernia compared with controls or those with indirect hernia. In contrast, patients with direct inguinal hernia showed significantly higher elastase expression. In fibroblasts isolated from the TF, relative amounts of tropoelastin mRNA were lower for the hernia groups but differences were not significant. LOXL-1 mRNA levels were significantly lower in the direct hernia group compared to controls. CONCLUSIONS: Our findings suggest that impaired elastic fibre function in the transversalis fascia of patients with direct inguinal hernia, reflected by diminished elastin synthesis and its enhanced enzyme degradation, contributes to the development of this type of hernia.

Down-regulation of lysyl oxydase-like in aging and venous insufficiency.

BACKGROUND: Elastin expression is higher in tissues where elastic fibres are essential for the correct maintenance of function such as blood vessels. Elastin expression usually diminishes with age, however, it may be re-expressed in response to injury or repair processes. Some authors attribute the characteristic loss of elasticity of the varicose vein to a drop in the population of smooth muscle cells in the media layer. A reduction in elastin has been observed in chronic venous insufficiency, but little is known about some of the factors involved in elastin synthesis such as lysyl oxidases. The aim of this study was to examine the in vivo expression of the elastin precursor, tropoelastin (TE), and lysyl oxidase-like 1 (LOXL1), a cross-linking enzyme responsible for elastin polymer deposition. The effects of age on these expression patterns were also evaluated. METHODS: Saphenous vein segments were obtained during surgery from organ donors (controls, n=20) and subjects with venous insufficiency (varicose veins, n=20). Both these groups were subdivided according to subject age into <50 years (n=10) and >or=50 years (n=10). Control and varicose vein tissue specimens were immunolabelled using anti-tropoelastin and anti-LOXL1 antibodies and also subjected to Western blot analysis. RESULTS: Our results indicate that the levels of these markers of elastin synthesis (LOXL/tropoelastin) in the vein wall diminish in a significant way (p<0.05) with the age factor. Excluding the age factor, LOXL1 was significantly decreased in the varicose condition (p<0.05). In the younger pathological population they showed an inverse relationship (LOXL decreased, tropoelastin increased). CONCLUSIONS: The already established reduction in elastin in the varicose condition may be related, at least in part, to the decreased LOXL1 levels observed here. These events could reduce spontaneous reticulation of elastin and the partial loss of tissue elasticity in this group of patients.

Age- and gender-related changes in ligament components.

The age- and gender-related changes in extracellular matrix components (elastin, elastin cross-links, fibrillin, collagen and glycoprotein) and mineral components (calcium, Ca; phosphorus, P) in human lumbar yellow ligaments were investigated using samples obtained from surgical specimens. The mineral (Ca and P) contents increased with ageing (r = 0.703 and r = 0.772, respectively), whereas the contents of matrix components tended to decrease with ageing (elastin r = -0.261, elastin cross-links r = -0.213, fibrillin r = 0.494; collagen r = -0.322 and glycoprotein r = -0.143). Comparison of the male and female groups revealed that the ligament elastin content and elastin cross-links decreased in the male group, whereas the ligament collagen content decreased in the female group significantly in an age-dependent manner (r = -0.788, r = -0.753 and r = -0.721, respectively). These findings demonstrate age- and gender-related changes in mineral and matrix components (especially elastin and collagen) in the lumbar yellow ligaments in the Japanese population. It is suggested that elastin and collagen metabolism in ligaments changes both with age and according to gender.

Posttranslational modifications of microfibril associated glycoprotein-1 (MAGP-1).

Microfibril-associated glycoprotein-1 (MAGP-1) is a small molecular weight protein associated with extracellular matrix microfibrils. Biochemical studies have shown that MAGP-1 undergoes several posttranslational modifications that may influence its associations with other microfibrillar components. To identify the sites in the molecule where posttranslational modifications occur, we expressed MAGP-1 constructs containing various point mutations as well as front and back half truncations in CHO cells. Characterization of transiently expressed protein showed that MAGP-1 undergoes O-linked glycosylation and tyrosine sulfation at sites in its amino-terminal half. This region of the protein also served as a major amine acceptor site for transglutaminase and mediated self-assembly into high molecular weight multimers through a glutamine-rich sequence. Fine mapping of the modification sites through mutational analysis demonstrated that Gln20 is a major amine acceptor site for the transglutaminase reaction and confirmed that a canonical tyrosine sulfation consensus sequence is the site of MAGP-1 sulfation. Our results also show that O-glycosylation occurs at more than one site in the molecule.

Overview of extracellular matrix.

The extracellular matrix provides an environment for cells. It is produced, assembled and modified by cells and in turn, it modifies the functions and behavior of the cells it encounters. The molecules that make up the matrix are diverse in both structure and function. This well-illustrated unit provides an introduction to the structure and function of the major components of matrix and serves as a background for the other units in the chapter which include protocols for isolation and analysis of individual components.

Revised genomic structure of the human MAGP1 gene and identification of alternate transcripts in human and mouse tissues.

The human MAGP1 (or MFAP2) and mouse Magp1 genes code for the microfibril-associated glycoprotein-1 (MAGP-1), an extracellular matrix protein of microfibrillar structures. We report a revised 5' genomic structure including the use of a single transcription start site that gives rise to a 32-bp 5' exon spanning a segment of the previously described exon B. No evidence of heterogeneous 5' ends from the use of alternative promoters was found in human tissues and cell lines. We located the genetic marker D1S170 to a position 3 kb downstream of the polyadenylation site. Large-scale comparison of the human and mouse genes revealed conservation of sequence outside the coding exons. Although the 5' flanking regions were found to be divergent certain cis-elements for transcription factors are conserved, including Sp1, AP-2, AP-4, NF-kappaB, and c-ETS motifs. We identified a total of five splice variants in addition to the canonical MAGP1A/Magp1A form. These transcripts are species-specific and are generated by different processing mechanisms. The alternate forms MAGP1A', MAGP1B, and MAGP1C are expressed in human tissues; and the two variants Magp1A" and Magp1D were found only in mouse. The alternatively spliced forms show restricted patterns of expression relative to the canonical isoform.

Mouse models of genetic diseases resulting from mutations in elastic fiber proteins.

The inability to study appropriate human tissues at various stages of development has precluded the elaboration of a thorough understanding of the pathogenic mechanisms leading to diseases linked to mutations in genes for elastic fiber proteins. Recently, new insights have been gained by studying mice harboring targeted mutations in the genes that encode fibrillin-1 and elastin. These genes have been linked to Marfan syndrome (MFS) and supravalvular aortic stenosis (SVAS), respectively. For fibrillin-1, mouse models have revealed that phenotype is determined by the degree of functional impairment. The haploinsufficiency state or the expression of low levels of a product with dominant-negative potential from one allele is associated with mild phenotypes with a predominance of skeletal features. Exuberant expression of a dominant-negative-acting protein leads to the more severe MFS phenotype. Mice harboring targeted deletion of the elastin gene (ELN) show many of the features of SVAS in humans, including abnormalities in the vascular wall and altered hemodynamics associated with changes in wall compliance. The genetically altered mice suggest that SVAS is predominantly a disease of haploinsufficiency. These studies have underscored the prominent role of the elastic matrix in the morphogenesis and homeostasis of the vessel wall.

Developmental expression of latent transforming growth factor beta binding protein 2 and its requirement early in mouse development.

Latent transforming growth factor beta (TGF-beta) binding protein 2 (LTBP-2) is an integral component of elastin-containing microfibrils. We studied the expression of LTBP-2 in the developing mouse and rat by in situ hybridization, using tropoelastin expression as a marker of tissues participating in elastic fiber formation. LTBP-2 colocalized with tropoelastin within the perichondrium, lung, dermis, large arterial vessels, epicardium, pericardium, and heart valves at various stages of rodent embryonic development. Both LTBP-2 and tropoelastin expression were seen throughout the lung parenchyma and within the cortex of the spleen in the young adult mouse. In the testes, LTBP-2 expression was seen within lumenal cells of the epididymis in the absence of tropoelastin. Collectively, these results imply that LTBP-2 plays a structural role within elastic fibers in most cases. To investigate its importance in development, mice with a targeted disruption of the Ltbp2 gene were generated. Ltbp2(-/-) mice die between embryonic day 3.5 (E3.5) and E6.5. LTBP-2 expression was not detected by in situ hybridization in E6.5 embryos but was detected in E3.5 blastocysts by reverse transcription-PCR. These results are not consistent with the phenotypes of TGF-beta knockout mice or mice with knockouts of other elastic fiber proteins, implying that LTBP-2 performs a yet undiscovered function in early development, perhaps in implantation.

The microfibrillar proteins MAGP-1 and fibrillin-1 form a ternary complex with the chondroitin sulfate proteoglycan decorin.

MAGP-1 and fibrillin-1, two protein components of extracellular microfibrils, were shown by immunoprecipitation studies to interact with the chondroitin sulfate proteoglycan decorin in the medium of cultured fetal bovine chondrocytes. Decorin interacted with each protein individually and with both proteins together to form a ternary complex. Expression of truncated fibrillin-1 proteins in Chinese hamster ovary cells localized proteoglycan binding to an amino-terminal region near the proline-rich domain. A spatially analogous fibrillin-2 truncated protein did not coprecipitate the same sulfated molecule, suggesting that chondroitin sulfate proteoglycan binding in this region is specific for fibrillin-1. An interaction between fibrillin and MAGP-1 was also observed under culture conditions that abrogated decorin secretion, suggesting that the two microfibrillar proteins can associate in the absence of the proteoglycan. Sulfation of matrix proteins is important for elastic fiber assembly because inhibition of sulfation was shown to prevent microfibrillar protein incorporation into the extracellular matrix of cultured cells.

Interaction of tropoelastin with the amino-terminal domains of fibrillin-1 and fibrillin-2 suggests a role for the fibrillins in elastic fiber assembly.

Alignment of tropoelastin molecules during the process of elastogenesis is thought to require fibrillin-containing microfibrils. In this study, we have demonstrated that amino-terminal domains of two microfibrillar proteins, fibrillin-1 and fibrillin-2, interact with tropoelastin in solid phase binding assays. The tropoelastin-binding site was localized to a region beginning at the glycine-rich and proline-rich regions of fibrillin-2 and fibrillin-1, respectively, and continuing through the second 8-cysteine domain. Characterization of the binding requirements using the fibrillin-2 construct found that a folded, secondary structure was necessary for binding. Furthermore, binding between tropoelastin and fibrillin was mediated by ionic interactions involving the lysine side chains of tropoelastin. The importance of the lysine side chains was corroborated by the finding that the fibrillin-2 construct did not bind to mature elastin, whose lysine side chains have been modified to form cross-links. Interestingly, there was no interaction between the fibrillin constructs and tropoelastin in solution phase, suggesting that binding of tropoelastin to a solid substrate exposes a cryptic binding site. These results suggest that fibrillin plays an important role in elastic fiber assembly by binding tropoelastin and perhaps facilitating side chain alignment for efficient cross-linking.

Relation between outer and luminal diameter in cannulated arteries.

Resistance in blood vessels is directly related to the inner (luminal) diameter (ID). However, ID can be difficult to measure during physiological experiments because of poor transillumination of thick-walled or tightly constricted vessels. We investigated whether the wall cross-sectional area (WCSA) in cannulated arteries is nearly constant, allowing IDs to be calculated from outer diameters (OD) using a single determination of WCSA. With the use of image analysis, OD and ID were directly measured using either transillumination or a fluorescent marker in the lumen. IDs from a variety of vessel types were calculated from WCSA at several reference pressures. Calculated IDs at all of the reference WCSA were within 5% (mean <1%) of the corresponding measured IDs in all vessel types studied, including vessels from heterozygote elastin knockout animals. This was true over a wide range of transmural pressures, during treatment with agonists, and before and after treatment with KCN. In conclusion, WCSA remains virtually constant in cannulated vessels, allowing accurate determination of ID from OD measurement under a variety of experimental conditions.

Characterization of an in vitro model of elastic fiber assembly.

Elastic fibers consist of two morphologically distinct components: elastin and 10-nm fibrillin-containing microfibrils. During development, the microfibrils form bundles that appear to act as a scaffold for the deposition, orientation, and assembly of tropoelastin monomers into an insoluble elastic fiber. Although microfibrils can assemble independent of elastin, tropoelastin monomers do not assemble without the presence of microfibrils. In the present study, immortalized ciliary body pigmented epithelial (PE) cells were investigated for their potential to serve as a cell culture model for elastic fiber assembly. Northern analysis showed that the PE cells express microfibril proteins but do not express tropoelastin. Immunofluorescence staining and electron microscopy confirmed that the microfibril proteins produced by the PE cells assemble into intact microfibrils. When the PE cells were transfected with a mammalian expression vector containing a bovine tropoelastin cDNA, the cells were found to express and secrete tropoelastin. Immunofluorescence and electron microscopic examination of the transfected PE cells showed the presence of elastic fibers in the matrix. Biochemical analysis of this matrix showed the presence of cross-links that are unique to mature insoluble elastin. Together, these results indicate that the PE cells provide a unique, stable in vitro system in which to study elastic fiber assembly.

An open reading frame element mediates posttranscriptional regulation of tropoelastin and responsiveness to transforming growth factor beta1.

Elastin, an extracellular component of arteries, lung, and skin, is produced during fetal and neonatal growth. We reported previously that the cessation of elastin production is controlled by a posttranscriptional mechanism. Although tropoelastin pre-mRNA is transcribed at the same rate in neonates and adults, marked instability of the fully processed transcript bars protein production in mature tissue. Using RNase protection, we identified a 10-nucleotide sequence in tropoelastin mRNA near the 5' end of the sequences coded by exon 30 that interacts specifically with a developmentally regulated cytosolic 50-kDa protein. Binding activity increased as tropoelastin expression dropped, being low in neonatal fibroblasts and high in adult cells, and treatment with transforming growth factor beta1 (TGF-beta1), which stimulates tropoelastin expression by stabilizing its mRNA, reduced mRNA-binding activity. No other region of tropoelastin mRNA interacted with cellular proteins, and no binding activity was detected in nuclear extracts. The ability of the exon-30 element to control mRNA decay and responsiveness to TGF-beta1 was assessed by three distinct functional assays: (i) insertion of exon 30 into a heterologous gene conferred increased reporter activity after exposure to TGF-beta1; (ii) addition of excess exon 30 RNA slowed tropoelastin mRNA decay in an in vitro polysome degradation assay; and (iii) a mutant tropoelastin cDNA lacking exon 30, compared to wild-type cDNA, produced a stable transcript whose levels were not affected by TGF-beta1. These findings demonstrate that posttranscriptional regulation of elastin production in mature tissue is conferred by a specific element within the open reading frame of tropoelastin mRNA.

N-terminal domains of fibrillin 1 and fibrillin 2 direct the formation of homodimers: a possible first step in microfibril assembly.

Aggregation of fibrillin molecules via disulphide bonds is postulated to be an early step in microfibril assembly. By expressing fragments of fibrillin 1 and fibrillin 2 in a mammalian expression system, we found that the N-terminal region of each protein directs the formation of homodimers and that disulphide bonds stabilize this interaction. A large fragment of fibrillin 1 containing much of the region downstream from the N-terminus remained as a monomer when expressed in the same cell system, indicating that this region of the protein lacks dimerization domains. This finding also confirms that the overexpression of fibrillin fragments does not in itself lead to spurious dimer formation. Pulse-chase analysis demonstrated that dimer formation occurred intracellularly, suggesting that the process of fibrillin aggregation is initiated early after biosynthesis of the molecules. These findings also implicate the N-terminal region of fibrillin 1 and fibrillin 2 in directing the formation of a dimer intermediate that aggregates to form the functional microfibril.

Processing of the fibrillin-1 carboxyl-terminal domain.

To investigate the processing and general properties of the fibrillin-1 carboxyl-terminal domain, three protein expression constructs have been developed as follows: one without the domain, one with the domain, and one with a mutation near the putative proteolytic processing site. The constructs have been expressed in two eukaryotic model systems, baculoviral and CHO-K1. Post-translational modifications that normally occur in fibrillin-1, including glycosylation, signal peptide cleavage, and carboxyl-terminal processing, occur in the three constructs in both cell systems. Amino-terminal sequencing of secreted protein revealed leader sequence processing at two sites, a primary site between Gly-24/Ala-25 and a secondary site of Ala-27/Asn-28. Processing of the carboxyl-terminal domain could be observed by migration differences in SDS-polyacrylamide gel electrophoresis and was evident in both mammalian and insect cells. Immunological identification by Western blotting confirmed the loss of the expected region. The failure of both cell systems to process the mutant construct shows that the multi-basic sequence is the site of proteolytic processing. Cleavage of the fibrillin-1 carboxyl-terminal domain occurred intracellularly in CHO-K1 cells in an early secretory pathway compartment as demonstrated by studies with secretion blocking agents. This finding, taken with the multi-basic nature of the cleavage site and observed calcium sensitivity of cleavage, suggests that the processing enzyme is a secretory pathway resident furin-like protease.