Cholinesterases: anchored enzymes in membranes and basal laminae

Two proteins, ColQ and PRiMA, organize tetramers of acetylcholinesterase (AChE) and of butyrylcholinesterase (BChE) through peptide interactions. A short proline rich sequence in the N-terminal domain of ColQ or PRiMA associates four C-terminal extension of AChE or BChE. ColQ targets the enzymes in the basal lamina, PRiMA targets the enzymes at the plasma membrane. These complexes represent the mature proteins. The unassembled C-terminal extention of AChE is the key determinant recognized during the "quality control" of protein synthesis. Unassembled catalytic subunits are then degraded by the proteasome pathway. At the neuromuscular junction, ColQ/AChE represents the concentrated enzyme. The clusterisation of AChE depends upon ColQ through three sites of interactions: two different heparin binding domains in the collagen domain interact with heparan sulfate proteoglycan particularly the perlecan and the C-terminal non collagenic domain interacts with MuSK, the tyrosine kinase receptor organiser of the neuromuscular junction. The absence of ColQ and AChE has revealed that the excess of Ach stimulates more nicotinic receptors but probably not until their desensitization. Several morphological modifications may help the clearance of Ach. Conversely the synapse transmission fails during high frequency nerve stimulation.     

The Cysteine-rich Region of Type VII Collagen Is a Cystine Knot with a New Topology

Collagens are a group of extracellular matrix proteins with essential functions for skin integrity. Anchoring fibrils are made of type VII collagen (Col7) and link different skin layers together: the basal lamina and the underlying connective tissue. Col7 has a central collagenous domain and two noncollagenous domains located at the N and C terminus (NC1 and NC2), respectively. A cysteine-rich region of hitherto unknown function is located at the transition of the NC1 domain to the collagenous domain. A synthetic model peptide of this region was investigated by CD and NMR spectroscopy. The peptide folds into a collagen triple helix, and the cysteine residues form disulfide bridges between the different strands. The eight cystine knot topologies that are characterized by exclusively intermolecular disulfide bridges have been analyzed by molecular modeling. Two cystine knots are energetically preferred; however, all eight disulfide bridge arrangements are essentially possible. This novel cystine knot is present in type IX collagen, too. The conserved motif of the cystine knot is CX₃CP. The cystine knot is N-terminal to the collagen triple helix in both collagens and therefore probably impedes unfolding of the collagen triple helix from the N terminus.     

Type XIII collagen forms homotrimers with three triple helical collagenous domains and its association into disulfide-bonded trimers is enhanced by prolyl 4-hydroxylase

Type XIII collagen is a type II transmembrane protein predicted to consist of a short cytosolic domain, a single transmembrane domain, and three collagenous domains flanked by noncollagenous sequences. Previous studies on mRNAs indicate that the structures of the collagenous domain closest to the cell membrane, COL1, the adjacent noncollagenous domain, NC2, and the C-terminal domains COL3 and NC4 are subject to alternative splicing. In order to extend studies of type XIII collagen from cDNAs to the protein level we have produced it in insect cells by means of baculoviruses. Type XIII collagen alpha chains were found to associate into disulfide-bonded trimers, and hydroxylation of proline residues dramatically enhanced this association. This protein contains altogether eight cysteine residues, and interchain disulfide bonds could be located in the NC1 domain and possibly at the junction of COL1 and NC2, while the two cysteine residues in NC4 are likely to form intrachain bonds. Pepsin and trypsin/chymotrypsin digestions indicated that the type XIII collagen alpha chains form homotrimers whose three collagenous domains are in triple helical conformation. The thermal stabilities (T(m)) of the COL1, COL2, and COL3 domains were 38, 49 and 40 degrees C, respectively. The T(m) of the central collagenous domain is unusually high, which in the light of this domain being invariant in terms of alternative splicing suggests that the central portion of the molecule may have an important role in the stability of the molecule. All in all, most of the type XIII collagen ectodomain appears to be present in triple helical conformation, which is in clear contrast to the short or highly interrupted triple helical domains of the other known collagenous transmembrane proteins.     

The origin of the molecular diversity and functional anchoring of cholinesterases

Vertebrates possess two cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) which both hydrolyze acetylcholine, but differ in their specificity towards other substrates, and in their sensitivity to inhibitors. In mammals, the AChE gene produces three types of coding regions through the choice of 3' splice acceptor sites, generating proteins which possess the same catalytic domain, associated with distinct C-terminal peptides. AChE subunits of type R ('readthrough') produce soluble monomers; they are expressed during development and induced by stress in the mouse brain. AChE subunits of type H ('hydrophobic') produce GPI-anchored dimers, but also secreted molecules; they are mostly expressed in blood cells. Subunits of type T ('tailed') exist for both AChE and BChE. They represent the enzyme forms expressed in brain and muscle. These subunits generate a variety of quaternary structures, including homomeric oligomers (monomers, dimers, tetramers), as well as hetero-oligomeric assemblies with anchoring proteins, ColQ and PRiMA. Mutations in the four-helix bundle (FHB) zone of the catalytic domain indicate that subunits of type H and T use the same interaction for dimerization. On the other hand, the C-terminal T peptide is necessary for tetramerization. Four T peptides, organized as amphiphilic alpha helices, can assemble around proline-rich motifs of ColQ or PRiMA. The association of AChE(T) or BChE subunits with ColQ produces collagen-tailed molecules, which are inserted in the extracellular matrix, e.g. in the basal lamina of neuromuscular junctions. Their association with PRiMA produces membrane-bound tetramers which constitute the predominant form of cholinesterases in the mammalian brain; in muscles, the level of PRiMA-anchored tetramers is regulated by exercise, but their functional significance remains unknown. In brain and muscles, the hydrolysis of acetylcholine by cholinesterases, in different contexts, and their possible noncatalytic functions clearly depend on their localization by ColQ or PRiMA.     

MuSK is required for anchoring acetylcholinesterase at the neuromuscular junction

At the neuromuscular junction, acetylcholinesterase (AChE) is mainly present as asymmetric forms in which tetramers of catalytic subunits are associated to a specific collagen, collagen Q (ColQ). The accumulation of the enzyme in the synaptic basal lamina strictly relies on ColQ. This has been shown to be mediated by interaction between ColQ and perlecan, which itself binds dystroglycan. Here, using transfected mutants of ColQ in a ColQ-deficient muscle cell line or COS-7 cells, we report that ColQ clusterizes through a more complex mechanism. This process requires two heparin-binding sites contained in the collagen domain as well as the COOH terminus of ColQ. Cross-linking and immunoprecipitation experiments in Torpedo postsynaptic membranes together with transfection experiments with muscle-specific kinase (MuSK) constructs in MuSK-deficient myotubes or COS-7 cells provide the first evidence that ColQ binds MuSK. Together, our data suggest that a ternary complex containing ColQ, perlecan, and MuSK is required for AChE clustering and support the notion that MuSK dictates AChE synaptic localization at the neuromuscular junction.     

Genetic analysis of collagen Q: roles in acetylcholinesterase and butyrylcholinesterase assembly and in synaptic structure and function

Acetylcholinesterase (AChE) occurs in both asymmetric forms, covalently associated with a collagenous subunit called Q (ColQ), and globular forms that may be either soluble or membrane associated. At the skeletal neuromuscular junction, asymmetric AChE is anchored to the basal lamina of the synaptic cleft, where it hydrolyzes acetylcholine to terminate synaptic transmission. AChE has also been hypothesized to play developmental roles in the nervous system, and ColQ is also expressed in some AChE-poor tissues. To seek roles of ColQ and AChE at synapses and elsewhere, we generated ColQ-deficient mutant mice. ColQ-/- mice completely lacked asymmetric AChE in skeletal and cardiac muscles and brain; they also lacked asymmetric forms of the AChE homologue, butyrylcholinesterase. Thus, products of the ColQ gene are required for assembly of all detectable asymmetric AChE and butyrylcholinesterase. Surprisingly, globular AChE tetramers were also absent from neonatal ColQ-/- muscles, suggesting a role for the ColQ gene in assembly or stabilization of AChE forms that do not themselves contain a collagenous subunit. Histochemical, immunohistochemical, toxicological, and electrophysiological assays all indicated absence of AChE at ColQ-/- neuromuscular junctions. Nonetheless, neuromuscular function was initially robust, demonstrating that AChE and ColQ do not play obligatory roles in early phases of synaptogenesis. Moreover, because acute inhibition of synaptic AChE is fatal to normal animals, there must be compensatory mechanisms in the mutant that allow the synapse to function in the chronic absence of AChE. One structural mechanism appears to be a partial ensheathment of nerve terminals by Schwann cells. Compensation was incomplete, however, as animals lacking ColQ and synaptic AChE failed to thrive and most died before they reached maturity.     

The synaptic acetylcholinesterase tetramer assembles around a polyproline II helix

Functional localization of acetylcholinesterase (AChE) in vertebrate muscle and brain depends on interaction of the tryptophan amphiphilic tetramerization (WAT) sequence, at the C-terminus of its major splice variant (T), with a proline-rich attachment domain (PRAD), of the anchoring proteins, collagenous (ColQ) and proline-rich membrane anchor. The crystal structure of the WAT/PRAD complex reveals a novel supercoil structure in which four parallel WAT chains form a left-handed superhelix around an antiparallel left-handed PRAD helix resembling polyproline II. The WAT coiled coils possess a WWW motif making repetitive hydrophobic stacking and hydrogen-bond interactions with the PRAD. The WAT chains are related by an approximately 4-fold screw axis around the PRAD. Each WAT makes similar but unique interactions, consistent with an asymmetric pattern of disulfide linkages between the AChE tetramer subunits and ColQ. The P59Q mutation in ColQ, which causes congenital endplate AChE deficiency, and is located within the PRAD, disrupts crucial WAT-WAT and WAT-PRAD interactions. A model is proposed for the synaptic AChE(T) tetramer.     

Acetylcholinesterase associates differently with its anchoring proteins ColQ and PRiMA

Acetylcholinesterase tetramers are inserted in the basal lamina of neuromuscular junctions or anchored in cell membranes through the interaction of four C-terminal t peptides with proline-rich attachment domains (PRADs) of cholinesterase-associated collagen Q (ColQ) or of the transmembrane protein PRiMA (proline-rich membrane anchor). ColQ and PRiMA differ in the length of their proline-rich motifs (10 and 15 residues, respectively). ColQ has two cysteines upstream of the PRAD, which are disulfide-linked to two AChE(T) subunits ("heavy" dimer), and the other two subunits are disulfide-linked together ("light" dimer). In contrast, PRiMA has four cysteines upstream of the PRAD. We examined whether these cysteines could be linked to AChE(T) subunits in complexes formed with PRiMA in transfected COS cells and in the mammalian brain. For comparison, we studied complexes formed with N-terminal fragments of ColQ, N-terminal fragments of PRiMA, and chimeras in which the upstream regions containing the cysteines were exchanged. We also compared the effect of mutations in the t peptides on their association with the two PRADs. We report that the two PRADs differ in their interaction with AChE(T) subunits; in complexes formed with the PRAD of PRiMA, we observed light dimers, but very few heavy dimers, even though such dimers were formed with the PQ chimera in which the N-terminal region of PRiMA was associated with the PRAD of ColQ. Complexes with PQ or with PRiMA contained heavy components, which migrated abnormally in SDS-PAGE but probably resulted from disulfide bonding of four AChE(T) subunits with the four upstream cysteines of the associated protein.     

Evidence for a type-IV-related collagen in Drosophila melanogaster. Evolutionary constancy of the carboxyl-terminal noncollagenous domain

Type IV collagen, a major structural component of basement membrane, has been characterized only in vertebrates. It is unique among the collagenous proteins in that it forms specific lattice networks by end-to-end interactions. In particular, in mammals the C-terminal noncollagenous domain (NCl) of collagen IV was shown to be one of the major cross-linking sites in the network assembly. Here, we give the first direct evidence of type-IV-related collagen in invertebrates by sequence analysis of cDNA and genomic DNA clones for the 3'-end of a previously characterized Drosophila collagen gene. The data describe the C-terminal 190 amino acid residues of the triple helix and the entire noncollagenous domain (231 amino acids) of the chain encoded for by this gene. Comparison with data reported for human and mouse alpha 1(IV) chains reveals that triple-helix regions are quite different, while NC1 structures are very similar. This suggests different constraints on triple-helix and NC1 domains during evolution. Present data support the assumption that the NC1 structure originated from duplication of an ancestral sequence; the extent of both interspecies and intramolecular homologies suggests the maintenance in vertebrates and invertebrates of an ancestral specific function.     

PRiMA: the membrane anchor of acetylcholinesterase in the brain.

As a tetramer, acetylcholinesterase (AChE) is anchored to the basal lamina of the neuromuscular junction and to the membrane of neuronal synapses. We have previously shown that collagen Q (ColQ) anchors AChE at the neuromuscular junction. We have now cloned the gene PRiMA (proline-rich membrane anchor) encoding the AChE anchor in mammalian brain. We show that PRiMA is able to organize AChE into tetramers and to anchor them at the surface of transfected cells. Furthermore, we demonstrate that AChE is actually anchored in neural cell membranes through its interaction with PRiMA. Finally, we propose that only PRiMA anchors AChE in mammalian brain and muscle cell membranes.     

Trimerization of collagen IX alpha-chains does not require the presence of the COL1 and NC1 domains

Collagen IX is a heterotrimer of three alpha-chains, which consists of three COL domains (collagenous domains) (COL1-COL3) and four NC domains (non-collagenous domains) (NC1-NC4), numbered from the C-terminus. Although collagen IX chains have been shown to associate via their C-terminal NC1 domains and form a triple helix starting from the COL1 domain, it is not known whether chain association can occur at other sites and whether other collagenous and non-collagenous regions are involved. To address this question, we prepared five constructs, two long variants (beginning at the NC4 domain) and three short variants (beginning at the COL2 domain), all ending at the NC2 domain (or NC2 replaced by NC1), to study association and selection of collagen IX alpha-chains. Both long variants were able to associate with NC1 or NC2 at the C-terminus and form various disulfide-bonded trimers, but the specificity of chain selection was diminished compared with full-length chains. Trimers of the long variant ending at NC2 were shown to be triple helical by CD. Short variants were not able to assemble into disulfide-bonded trimers even in the presence of both conserved cysteine residues from the COL1-NC1 junction. Our results demonstrate that collagen IX alpha-chains can associate in the absence of COL1 and NC1 domains to form a triple helix, but the COL2-NC2 region alone is not sufficient for trimerization. The results suggest that folding of collagen IX is a co-operative process involving multiple COL and NC domains and that the COL1-NC1 region is important for chain specificity.     

COOH-terminal collagen Q (COLQ) mutants causing human deficiency of endplate acetylcholinesterase impair the interaction of ColQ with proteins of the basal lamina

Collagen Q (ColQ) is a key multidomain functional protein of the neuromuscular junction (NMJ), crucial for anchoring acetylcholinesterase (AChE) to the basal lamina (BL) and accumulating AChE at the NMJ. The attachment of AChE to the BL is primarily accomplished by the binding of the ColQ collagen domain to the heparan sulfate proteoglycan perlecan and the COOH-terminus to the muscle-specific receptor tyrosine kinase (MuSK), which in turn plays a fundamental role in the development and maintenance of the NMJ. Yet, the precise mechanism by which ColQ anchors AChE at the NMJ remains unknown. We identified five novel mutations at the COOH-terminus of ColQ in seven patients from five families affected with endplate (EP) AChE deficiency. We found that the mutations do not affect the assembly of ColQ with AChE to form asymmetric forms of AChE or impair the interaction of ColQ with perlecan. By contrast, all mutations impair in varied degree the interaction of ColQ with MuSK as well as basement membrane extract (BME) that have no detectable MuSK. Our data confirm that the interaction of ColQ to perlecan and MuSK is crucial for anchoring AChE to the NMJ. In addition, the identified COOH-terminal mutants not only reduce the interaction of ColQ with MuSK, but also diminish the interaction of ColQ with BME. These findings suggest that the impaired attachment of COOH-terminal mutants causing EP AChE deficiency is in part independent of MuSK, and that the COOH-terminus of ColQ may interact with other proteins at the BL.     

Molecular modeling of the collagen-like tail of asymmetric acetylcholinesterase

The asymmetric form of acetylcholinesterase comprises three catalytic tetramers attached to ColQ, a collagen-like tail responsible for the anchorage of the enzyme to the synaptic basal lamina. ColQ is composed of an N-terminal domain which interacts with the catalytic subunits of the enzyme, a central collagen-like domain and a C-terminal globular domain. In particular, the collagen-like domain of ColQ contains two heparin-binding domains which interact with heparan sulfate proteoglycans in the basal lamina. A three-dimensional model of the collagen-like domain of the tail of asymmetric acetylcholinesterase was constructed. The model presents an undulated shape that results from the presence of a substitution and an insertion in the Gly-X-Y repeating pattern, as well as from low imino-acid regions. Moreover, this model permits the analysis of interactions between the heparin-binding domains of ColQ and heparin, and could also prove useful in the prediction of interaction domains with other putative basal lamina receptors.     

Reciprocal neural regulation of extrajunctional acetylcholinesterase and collagen Q in rat muscles—The role of calcineurin signaling

There are two main differences regarding acetylcholinesterase (AChE) expression in the extrajunctional regions of fast and slow rat muscles: (1) the activity of AChE catalytic subunits (G1 form) is much higher in fast than in slow muscles, and (2) the activity of the asymmetric forms of AChE (A₈ and A₁₂) is quite high extrajunctionally in slow muscles but virtually absent in fast muscles. The latter is due to the absence of the expression of AChE-associated collagen Q (ColQ) in the extrajunctional regions of fast muscle fibers, in contrast to its ample expression in slow muscles. We showed that both differences are caused by different neural activation patterns of fast vs. slow muscle fibers, which determine the respective levels of mRNA of both proteins. Whereas the changes in AChE mRNA levels in fast and slow muscles, as well as the levels of ColQ mRNA levels in slow muscles, observed in response to exposing either slow or fast muscles to different muscle activation patterns, are completely reversible, the extrajunctional suppression of ColQ expression in fast muscle fibers seems to be irreversible. Calcineurin signaling pathway in muscles is activated by high-average sarcoplasmic calcium concentration resulting from tonic low-frequency muscle fiber activation pattern, typical for slow muscle fibers, but is inactive in fast muscle fibers, which are activated by infrequent high-frequency bursts of neural impulses. Application to rats of two inhibitors of calcineurin (tacrolimus-FK506 and cyclosporin A) demonstrated that the mRNA levels of both the AChE catalytic subunit and ColQ in the extrajunctional regions of the soleus muscle are regulated by the calcineurin signaling pathway, but in a reciprocal way. Under the conditions of low calcineurin activity, AChE expression is enhanced and that of ColQ is suppressed, and vice versa. Our results also indicated that different, calcineurin-independent regulatory pathways are responsible for the reduction of AChE expression during muscle denervation, and for maintaining high ColQ expression in the neuromuscular junctions of fast muscle fibers.     

The alpha 1 chain of type XIII collagen consists of three collagenous and four noncollagenous domains, and its primary transcript undergoes complex alternative splicing.

We have recently reported a characterization of cDNA clones that encode an apparently novel human collagen that undergoes alternative splicing. These cDNAs covered one-third of the corresponding 2.5-2.8-kilobase mRNAs. We have now determined the complete primary structure of the protein encoded by several overlapping cDNAs isolated from a human endothelial cell library. Since the deduced translation product of the cDNAs is different in structure from all other collagen types, we have given the collagen chain encoded by the cDNAs the designation alpha 1 (XIII). The deduced polypeptide consists of three collagenous domains and four noncollagenous domains, two of them separating the collagenous domains and two located at the N-terminal and C-terminal ends of the polypeptide. Cysteine residues are found in three of the noncollagenous domains and also in the extreme N-terminal collagenous domain. Surprisingly, comparison of the nucleotide sequences encoded by the overlapping cDNA clones demonstrates that there are several alpha 1 (XIII) collagen mRNAs in HT-1080 human fibrosarcoma cells and human endothelial cells which differ in coding potential. Nuclease S1 mapping experiments suggest that these different mRNAs arise through alternative splicing of the precursor RNA at five locations within the coding region. This property makes type XIII collagen unique among all the collagen types studied so far. Its polypeptide length, therefore, may vary between 614 and 526 amino acids, depending on what internal splicing has taken place.     

Acetylcholinesterase: C-terminal domains,molecular forms and functional localization

Acetylcholinesterase (AChE) possesses short C-terminal peptides that are not necessary for catalytic activity. These peptides belong to different classes (R, H, T, S) and define the post-translational processing and targeting of the enzyme. In vertebrates, subunits of type H (AChE_H) and of type T (AChE_T) are the most important: AChE_H subunits produced glycolipid (GPI)-anchored dimers and AChE_T subunits produce hetero-oligometric forms such as membrane-bound tetramer in the mammalian brain (containing a 20 kDa hydrophobic protein) and asymmetric collagen-tailed forms in neuromuscular junctions (containing a specific collagen, ColQ). The T peptide allows the formation of tetrameric assemblies with a proline-rich attachment domain (PRAD) of collagen ColQ. These complex molecular structures condition the functional localization of the enzyme in the supramolecular architecture of cholinegic synapses.     

Complete primary structure of human collagen alpha 1 (V) chain

Several cDNA clones, encoding prepropeptide of human collagen alpha 1(V) chain, have been isolated. The prepropeptide (1838 amino acids length) of the alpha 1(V) chain was composed of a putative signal peptide, a large NH2-terminal noncollagenous region, a main collagenous region, and a COOH-terminal noncollagenous region. The signal peptide contained many leucine residues. The NH2-terminal noncollagenous region was much larger than those of the other collagens and had a region homologous to the COOH-terminal domain of laminin A chain, but it did not contain a cysteine-rich region that was maintained in the region of the other collagens. This region also contained probable tyrosine sulfation sites, and short collagenous sequences that were interrupted by three noncollagenous segments. The main collagenous region of the alpha 1(V) chain consisted of 338 repeats of Gly-X-Y-triplet. This region had a high degree (82%) of homology with the amino acids of the collagen alpha 1(XI) chain. The COOH-terminal noncollagenous region resembled that of the alpha 1(XI) chain, too, and 8 residues of cysteine that were important for the formation of the triple helix structure of collagens were observed. These results suggest that the alpha 1(V) chain belongs to the fibrillar collagen relative to the alpha 1(XI) chain, but codon usage of the alpha 1(V) cDNA was clearly different from those of the other fibrillar collagens including the alpha 1(XI), while it was similar to type IV collagen. This result supposes a different evolution of the alpha 1(V) gene from those of the other fibrillar collagens.     

Elements of the C-terminal t peptide of acetylcholinesterase that determine amphiphilicity, homomeric and heteromeric associations, secretion and degradation.

The C-terminal t peptide (40 residues) of vertebrate acetylcholinesterase (AChE) T subunits possesses a series of seven conserved aromatic residues and forms an amphiphilic alpha-helix; it allows the formation of homo-oligomers (monomers, dimers and tetramers) and heteromeric associations with the anchoring proteins, ColQ and PRiMA, which contain a proline-rich motif (PRAD). We analyzed the influence of mutations in the t peptide of Torpedo AChE(T) on oligomerization and secretion. Charged residues influenced the distribution of homo-oligomers but had little effect on the heteromeric association with Q(N), a PRAD-containing N-terminal fragment of ColQ. The formation of homo-tetramers and Q(N)-linked tetramers required a central core of four aromatic residues and a peptide segment extending to residue 31; the last nine residues (32-40) were not necessary, although the formation of disulfide bonds by cysteine C37 stabilized T(4) and T(4)-Q(N) tetramers. The last two residues of the t peptide (EL) induced a partial intracellular retention; replacement of the C-terminal CAEL tetrapeptide by KDEL did not prevent tetramerization and heteromeric association with Q(N), indicating that these associations take place in the endoplasmic reticulum. Mutations that disorganize the alpha-helical structure of the t peptide were found to enhance degradation. Co-expression with Q(N) generally increased secretion, mostly as T(4)-Q(N) complexes, but reduced it for some mutants. Thus, mutations in this small, autonomous interaction domain bring information on the features that determine oligomeric associations of AChE(T) subunits and the choice between secretion and degradation.     

The C5 domain of the collagen VI alpha3(VI) chain is critical for extracellular microfibril formation and is present in the extracellular matrix of cultured cells

Collagen VI, a microfibrillar protein found in virtually all connective tissues, is composed of three distinct subunits, alpha1(VI), alpha2(VI), and alpha3(VI), which associate intracellularly to form triple helical heterotrimeric monomers then dimers and tetramers. The secreted tetramers associate end-to-end to form beaded microfibrils. Although the basic steps in assembly and the structure of the tetramers and microfibrils are well defined, details of the interacting protein domains involved in assembly are still poorly understood. To explore the role of the C-terminal globular regions in assembly, alpha3(VI) cDNA expression constructs with C-terminal truncations were stably transfected into SaOS-2 cells. Control alpha3(VI) N6-C5 chains with an intact C-terminal globular region (subdomains C1-C5), and truncated alpha3(VI) N6-C1, N6-C2, N6-C3, and N6-C4 chains, all associated with endogenous alpha1(VI) and alpha2(VI) to form collagen VI monomers, dimers and tetramers, which were secreted. These data demonstrate that subdomains C2-C5 are not required for monomer, dimer or tetramer assembly, and suggest that the important chain selection interactions involve the C1 subdomains. In contrast to tetramers containing control alpha3(VI) N6-C5 chains, tetramers containing truncated alpha3(VI) chains were unable to associate efficiently end-to-end in the medium and did not form a significant extracellular matrix, demonstrating that the alpha3(VI) C5 domain plays a crucial role in collagen VI microfibril assembly. The alpha3(VI) C5 domain is present in the extracellular matrix of SaOS-2 N6-C5 expressing cells and fibroblasts demonstrating that processing of the C-terminal region of the alpha3(VI) chain is not essential for microfibril formation.