Carbamylated albumin is a potent inhibitor of polymorphonuclear neutrophil respiratory burst

Carbamylation is a post-translational modification of proteins characterized by the binding of cyanate to amino groups, increased in renal failure. Pathophysiological consequences of carbamylation and adverse effects of carbamylated proteins on cell functions are poorly understood. We studied the influence of carbamylated albumin on polymorphonuclear neutrophil (PMN) O(2)(-) production. Carbamylated albumin significantly decreased O(2)(-) production in PMNs stimulated by type I collagen, but not by phorbol 12-myristate 13-acetate or tumor necrosis factor-alpha. This effect was related to inhibition of p(125)FAK phosphorylation. Such an alteration of neutrophil oxidative functions might explain characteristic complications of renal failure, such as increased occurrence of inflammation or infections.     

Enhanced activation of and increased production of matrix metalloproteinase-9 by human blood monocytes upon adhering to carbamylated collagen

Carbamylation refers to chemical modification of protein side chains by cyanate derived e.g. from urea. It alters their structural and functional properties. We have studied the influence of the carbamylation of type I collagen in vitro on its interactions with elutriated human monocytes, and its potential role in atherosclerosis. Adhesion of monocytes onto carbamylated collagen was significantly enhanced compared to native collagen. There was no change in superoxide anion production. On the other hand, there was an increase in the production and the activation of matrix metalloproteinase-9. No effect was found on tissue inhibitor of metalloproteinase-1 production. Thus, the presence of carbamylated collagen may stimulate the remodelling of extracellular matrix mediated by activated monocytes. Such alterations may contribute to enhanced atherosclerosis in renal insufficiency, a pathological condition associated with elevated levels of carbamylation.     

Involvement of lysine 1047 in type I collagen-mediated activation of polymorphonuclear neutrophils

Oxidative functions of polymorphonuclear neutrophils (PMNs), which play a deciding role in the phagocytosis process, are stimulated by extracellular matrix proteins such as type I collagen. Previous studies have demonstrated the involvement of a DGGRYY sequence located within the alpha(1) chain C-terminal telopeptide in type I collagen-induced PMN activation, but so far the mechanism has not been completely elucidated. We have recently demonstrated that collagen carbamylation (i.e. post-translational binding of cyanate to lysine epsilon-NH(2) groups) impairs PMN oxidative functions, suggesting the potential involvement of lysine residues in this process. The present study was devoted to the identification of lysine residues involved in the collagen-induced activation of PMNs. The inhibition of PMN activation by collagen in the presence of 6-amino-hexanoic acid, a structural analogue of lysine residues, confirmed the involvement of specific lysine residues. Modification of lysine residues by carbamylation demonstrated that only one residue, located within the alpha(1)CB6 collagen peptide, was involved in this mechanism. A recombinant alpha(1)CB6 peptide, designed for the substitution of lysine 1047 by glycine, exhibited decreased activity, demonstrating that the lysine residue at position 1047 within the collagen molecule played a significant role in the mechanism of activation. These results help to understand in more detail the collagen-mediated PMN activation mechanism and confirm the prominent involvement of lysine residues in interactions between extracellular matrix proteins and inflammatory cells.     

In vivo carbamylation and acetylation of water-soluble human lens alphaB-crystallin lysine 92

Several post-translational modifications of lysine residues of lens proteins have been implicated in cataractogenesis. In the present study, the molecular weight of an alpha-crystallin isolated from the water-soluble portion of a cataractous human eye lens indicated that it was a modified alphaB-crystallin. Further analysis by mass spectrometry of tryptic digests of this modified protein showed that Lys 92 was modified and that the sample was structurally heterogeneous. Lys 92 was acetylated in one population and carbamylated in another. Although carbamylation of lens crystallins has been predicted, this is the first documentation of in vivo carbamylation of a specific site. These results are also the first documentation of in vivo lysine acetylation of alphaB-crystallin. Both modifications alter the net charge on alphaB-crystallin, a feature that may have significance to cataractogenesis.     

Endogenous Carbamylation of Renal Medullary Proteins

Protein carbamylation is a post-translational modification that can occur in the presence of urea. In solution, urea is in equilibrium with ammonium cyanate, and carbamylation occurs when cyanate ions react with the amino groups of lysines, arginines, protein N-termini, as well as sulfhydryl groups of cysteines. The concentration of urea is elevated in the renal inner medulla compared with other tissues. Due to the high urea concentration, we hypothesized that carbamylation can occur endogenously within the rat inner medulla. Using immunoblotting of rat kidney cortical and medullary homogenates with a carbamyl-lysine specific antibody, we showed that carbamylation is present in a large number of inner medullary proteins. Using protein mass spectrometry (LC-MS/MS) of rat renal inner medulla, we identified 456 unique carbamylated sites in 403 proteins, including many that play important physiological roles in the renal medulla [Data can be accessed at https://helixweb.nih.gov/ESBL/Database/Carbamylation/Carbamylation_peptide_sorted.html]. We conclude that protein carbamylation occurs endogenously in the kidney, modifying many physiologically important proteins.     

Carbamylation of alkaline mesentericopeptidas.

The effects of carbamylation with potassium cyanate, and methylation with methyl p-nitrobenzene sulphonate on the mesentericopeptidase activity are studies. The treatment with potassium cyanate causes the enzyme to lose its activity towards ester substrates and casein. The specific reagent N-trans-cinnamoylimidazole does not acylate the active site in the carbamylated enzyme. The pH dependence of the rate of inactivation indicates that an ionizing group of pK = 7.3, probably the protonated imidazole group of the active site histidine, is involved in the reaction. The competitive inhibitor boric acid protects mesentericopeptidase against inactivation with potassium cyanate. These suggest that the active site residues are modified in the unprotected enzyme. Sixty per cent of the enzyme activity toward N-acetyl-L-tyrosine ethyl ester was restored after treatment of the carbamylated mesentericopeptidase with 1 M hydroxylamine hydrochloride. Circular dichroism spectra show that the carbamylation does not change markedly the native protein conformation.     

Fructose 1,6-bisphosphatase: Dissociation of AMP cooperativity and AMP inhibition by carbamylation

Selective treatment of pig kidney fructose 1,6-bisphosphatase with potassium cyanate leads to the formation of an active carbamylated enzyme that has lost the cooperative interactions among AMP sites, but retains sensitivity to inhibition of catalytic activity by the regulator AMP. Incorporation data on [¹⁴C]KNCO indicate that the loss of enzyme cooperativity at the AMP sites is related to selective carbamylation of four lysine residues per mole of tetrameric enzyme. Exhaustive carbamylation suggests that a second lysine residue per subunit is essential for AMP inhibition.     

Evidence of in situ stability of the type IV collagen triple helix in human inflammatory bowel disease using a denaturation specific epitope antibody.

A peptide specific antibody (AH1OW1) was raised against an epitope, AH10 (aa 449-463), of the alpha1(IV) chain adjacent to a cleavage site for matrix metalloproteinases (MMP)-2 and -9 within the triple helix of type IV collagen. The antibody only reacted with denatured and reduced preparations of type IV collagen, or with pepsin isolated type IV collagen digested with MMP-2 and MMP-9. The specificity of this antibody for the denatured triple helix was demonstrated by the lack of staining with pre-immune antibody and by pre-incubation of AH1OW1 antibody with excess AH10 peptide epitope. The AH1OWI antibody was used to detect whether proteolysis of type IV collagen occurs in ulcerative colitis, an inflammatory bowel condition often characterised by a large influx of granulocytes and macrophages and an associated tissue destruction. However, no evidence of in situ proteolysis of the basement membrane type IV collagen was observed. Only in the most actively inflamed mucosa was staining with AH1OW1 antibody observed in the mucosal connective tissue. Digestion of frozen sections of bowel with MMP-1, MMP-2, MMP-3 and MMP-9 did not result in the exposure of the AH10 epitope. These data demonstrate the stability of intact type IV collagen and indicate that susceptibility of alpha1(IV) chain to digestion with MMP-2 and MMP-9 may require other proteolytic/denaturing events in the molecule.     

Production of recombinant human type I procollagen trimers using a four-gene expression system in the yeast Saccharomyces cerevisiae

The expression of stable recombinant human collagen requires an expression system capable of post-translational modifications and assembly of the procollagen polypeptides. Two genes were expressed in the yeast Saccharomyces cerevisiae to produce both propeptide chains that constitute human type I procollagen. Two additional genes were expressed coding for the subunits of prolyl hydroxylase, an enzyme that post-translationally modifies procollagen and that confers heat (thermal) stability to the triple helical conformation of the collagen molecule. Type I procollagen was produced as a stable heterotrimeric helix similar to type I procollagen produced in tissue culture. A key requirement for glutamate was identified as a medium supplement to obtain high expression levels of type I procollagen as heat-stable heterotrimers in Saccharomyces. Expression of these four genes was sufficient for correct assembly and processing of type I procollagen in a eucaryotic system that does not produce collagen.     

Characterization of the mechanisms by which gelatinase A, neutrophil collagenase, and membrane-type metalloproteinase MMP-14 recognize collagen I and enzymatically process the two alpha-chains

The turnover of native collagen has been ascribed to different members of the matrix metalloproteinase (MMP) family. Here, the mechanisms by which neutrophil collagenase (MMP-8), gelatinase A (MMP-2), and the ectodomain of MT1-MMP (ectMMP-14) degrade fibrillar collagen were examined. In particular, the hydrolysis of type I collagen at 37 degrees C was investigated to identify functional differences in the processing of the two alpha-chain types of fibrillar collagen. Thermodynamic and kinetic parameters were used for a quantitative comparison of the binding, unwinding, and hydrolysis of triple helical collagen. We demonstrate that the MMP family has developed at least two distinct mechanisms for collagen unwinding and cleavage. MMP-8 and ectMMP-14 display a similar mechanism (although with different catalytic parameters), which is characterized by binding (likely through the hemopexin-like domain) and cleavage of alpha-1 and/or alpha-2 chains without distinguishing between them and keeping the gross conformation of the triple helix (at least during the first cleavage step). On the other hand, MMP-2 binds preferentially the alpha-1 chains (likely through the fibronectin-like domain, which is not present in MMP-8 and ectMMP-14), grossly altering the whole triple helical arrangement of the collagen molecule and cleaving preferentially the alpha-2 chain. These distinctive mechanisms underly a drastically different mode of interaction with triple helical fibrillar collagen I, according to which the MMP domain is involved in binding. These findings can be related to the different role exerted by these MMPs on collagen homeostasis in the extracellular matrix.     

Characterization of the distinct collagen binding, helicase and cleavage mechanisms of matrix metalloproteinase 2 and 14 (gelatinase A and MT1-MMP): the differential roles of the MMP hemopexin c domains and the MMP-2 fibronectin type II modules in collagen triple helicase activities

Matrix metalloproteinase-2 (MMP-2, gelatinase A) and membrane type (MT)1-MMP (MMP-14) are cooperative dynamic components of a cell surface proteolytic axis involved in regulating the cellular signaling environment and pericellular collagen homeostasis. Although MT1-MMP exhibits type I collagenolytic but poor gelatinolytic activities, MMP-2 is a potent gelatinase with weak type I collagenolytic behavior. Recombinant linker/hemopexin C domain (LCD) of MT1-MMP binds native type I collagen, blocks MT1-MMP collagenolytic activity in trans, and by circular dichroism spectroscopy, induces localized structural perturbation in the collagen. These changes were reflected by enhanced cleavage of the MT1-LCD-bound collagen by the collagenases MMP-1 and MMP-8 but not by trypsin or MMP-7. Thus, the MT1-LCD alone can initiate triple helicase activity. In contrast, the native and denatured collagen binding properties of MMP-2 reside in the fibronectin type II modules, accordingly termed the collagen binding domain (CBD). Recombinant CBD (but not the MMP-2 LCD) also changed the circular dichroism spectra leading to increased MMP-1 and -8 cleavage of native collagen. However, recombinant CBD reduced gelatin and collagen cleavage by MMP-2 in trans as did CBD23, which comprises the second and third fibronectin type II modules, but not the CBD23 mutant W316A/W374A, which neither binds gelatin nor collagen. This indicates that MMP-2 and MT1-MMP bind collagen at a different site than MMP-1 and MMP-8. Thus, MMP-2 utilizes the CBD in cis for collagen binding and triple helicase activity, which compensates for the lack of collagen binding by the MMP-2 LCD. Hence, the MMP family has evolved two distinct mechanisms for collagen triple helicase activity using two structurally distinct domains, with triple helicase activity occurring independent of alpha-chain hydrolysis.     

Generation of glycosylated remnant epitopes from human collagen type II by gelatinase B

Gelatinase B/matrix metalloproteinase-9 (MMP-9) is an inflammatory mediator and effector. Considerable amounts of gelatinase B are released by neutrophils in the synovial cavity of patients with rheumatoid arthritis, and gelatinase B-deficient mice are resistant against antibody-induced arthritis. Native human collagen type II is susceptible to cleavage by various collagenases (MMP-1, MMP-8, and MMP-13), which cleave at a single position in the triple helix. Although the triple-helical structure may persist after this single cleavage, we show that gelatinase B degrades the resulting fragments into small remnant peptides. These were identified by mass spectrometry and Edman degradation. Localization of 31 cleavage sites shows that the immunodominant epitopes remain intact after cleavage and may become available, processed as antigens and presented in MHC-II molecules. Furthermore, most post-translational modifications were identified on the fragments, including nine glycosylation sites. In particular, it is shown for the first time by structural analysis that in natural human collagen II, lysines in the main immunodominant epitope are modified by partial hydroxylation and partial glycosylation. Determination of T-cell reactivity against such fragments indicates that, besides the two known main immunodominant epitopes, other glyco-epitopes may be present in collagen II. This reinforces the role of glycopeptide antigens in autoimmunity.     

The roles of substrate thermal stability and P2 and P1' subsite identity on matrix metalloproteinase triple-helical peptidase activity and collagen specificity

The hydrolysis of collagen (collagenolysis) is one of the committed steps in extracellular matrix turnover. Within the matrix metalloproteinase (MMP) family distinct preferences for collagen types are seen. The substrate determinants that may guide these specificities are unknown. In this study, we have utilized 12 triple-helical substrates in combination with 10 MMPs to better define the contributions of substrate sequence and thermal stability toward triple helicase activity and collagen specificity. In general, MMP-13 was found to be distinct from MMP-8 and MT1-MMP(Delta279-523), in that enhanced substrate thermal stability has only a modest effect on activity, regardless of sequence. This result correlates to the unique collagen specificity of MMP-13 compared with MMP-8 and MT1-MMP, in that MMP-13 hydrolyzes type II collagen efficiently, whereas MMP-8 and MT1-MMP are similar in their preference for type I collagen. In turn, MMP-1 was the least efficient of the collagenolytic MMPs at processing increasingly thermal stable triple helices and thus favors type III collagen, which has a relatively flexible cleavage site. Gelatinases (MMP-2 and MMP-9(Delta444-707)) appear incapable of processing more stable helices and are thus mechanistically distinct from collagenolytic MMPs. The collagen specificity of MMPs appears to be based on a combination of substrate sequence and thermal stability. Analysis of the hydrolysis of triple-helical peptides by an MMP mutant indicated that Tyr(210) functions in triple helix binding and hydrolysis, but not in processing triple helices of increasing thermal stabilities. Further exploration of MMP active sites and exosites, in combination with substrate conformation, may prove valuable for additional dissection of collagenolysis and yield information useful in the design of more selective MMP inhibitors.     

Mimivirus collagen is modified by bifunctional lysyl hydroxylase and glycosyltransferase enzyme

Collagens, the most abundant proteins in animals, are modified by hydroxylation of proline and lysine residues and by glycosylation of hydroxylysine. Dedicated prolyl hydroxylase, lysyl hydroxylase, and collagen glycosyltransferase enzymes localized in the endoplasmic reticulum mediate these modifications prior to the formation of the collagen triple helix. Whereas collagen-like proteins have been described in some fungi, bacteria, and viruses, the post-translational machinery modifying collagens has never been described outside of animals. We demonstrate that the L230 open reading frame of the giant virus Acanthamoeba polyphaga mimivirus encodes an enzyme that has distinct lysyl hydroxylase and collagen glycosyltransferase domains. We show that mimivirus L230 is capable of hydroxylating lysine and glycosylating the resulting hydroxylysine residues in a native mimivirus collagen acceptor substrate. Whereas in animals from sponges to humans the transfer of galactose to hydroxylysine in collagen is conserved, the mimivirus L230 enzyme transfers glucose to hydroxylysine, thereby defining a novel type of collagen glycosylation in nature. The presence of hydroxylysine in mimivirus proteins was confirmed by amino acid analysis of mimivirus recovered from A. polyphaga cultures. This work shows for the first time that collagen post-translational modifications are not confined to the domains of life. The utilization of glucose instead of the galactose found throughout animals as well as a bifunctional enzyme rather than two separate enzymes may represent a parallel evolutionary track in collagen biology. These results suggest that giant viruses may have contributed to the evolution of collagen biology.     

Abnormal type I collagen metabolism by cultured fibroblasts in lethal perinatal osteogenesis imperfecta.

Cultured skin fibroblasts from seven consecutive cases of lethal perinatal osteogenesis imperfecta (OI) expressed defects of type I collagen metabolism. The secretion of [14C]proline-labelled collagen by the OI cells was specifically reduced (51-79% of control), and collagen degradation was increased to twice that of control cells in five cases and increased by approx. 30% in the other two cases. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that four of the OI cell lines produced two forms of type I collagen consisting of both normally and slowly migrating forms of the alpha 1(I)- and alpha 2(I)-chains. In the other three OI cell lines only the 'slow' alpha (I)'- and alpha 2(I)'-chains were detected. In both groups inhibition of the post-translational modifications of proline and lysine resulted in the production of a single species of type I collagen with normal electrophoretic migration. Proline hydroxylation was normal, but the hydroxylysine contents of alpha 1(I)'- and alpha 2(I)'-chains purified by h.p.l.c. were greater than in control alpha-chains. The glucosylgalactosylhydroxylysine content was increased approx. 3-fold while the galactosylhydroxylysine content was only slightly increased in the alpha 1(I)'-chains relative to control alpha 1(I)-chains. Peptide mapping of the CNBr-cleavage peptides provided evidence that the increased post-translational modifications were distributed throughout the alpha 1(I)'- and alpha 2(I)'-chains. It is postulated that the greater modification of these chains was due to structural defects of the alpha-chains leading to delayed helix formation. The abnormal charge heterogeneity observed in the alpha 1 CB8 peptide of one patient may reflect such a structural defect in the type I collagen molecule.     

Substitutions for glycine alpha 1-637 and glycine alpha 2-694 of type I procollagen in lethal osteogenesis imperfecta. The conformational strain on the triple helix introduced by a glycine substitution can be transmitted along the helix

Two substitutions for glycine in the triple-helical domain were found in type I procollagen synthesized by skin fibroblasts from two probands with lethal osteogenesis imperfecta. One was a substitution of valine for glycine alpha 1-637, and the other was a substitution of arginine for glycine alpha 2-694. The effects of the mutations on the zipper-like folding of the collagen triple helix were similar, since there was post-translational overmodification of the collagenase A fragments (amino acids 1-775) but not of more COOH-terminal fragments of the protein. The mutations differed markedly, however, on their effects on thermal unfolding of the triple helix. The collagenase A fragment from the collagen containing the arginine alpha 2-694 substitution was cleaved at about amino acid 700 when incubated with trypsin at 30-35 degrees C. Therefore, there was micro-unfolding of the triple helix at a site close to the glycine substitution. Surprisingly, however, the collagenase A fragment with the valine alpha 1-637 substitution was also cleaved at about amino acid 700 under the same conditions. The results, therefore, demonstrated that although most glycine substitutions delay folding of the triple helix in regions that are NH2-terminal to the site of the substitution, the effects on unfolding can be transmitted to regions that are COOH-terminal to the site of the glycine substitution.     

Defining requirements for collagenase cleavage in collagen type III using a bacterial collagen system

Degradation of fibrillar collagens is important in many physiological and pathological events. These collagens are resistant to most proteases due to the tightly packed triple-helical structure, but are readily cleaved at a specific site by collagenases, selected members of the matrix metalloproteinases (MMPs). To investigate the structural requirements for collagenolysis, varying numbers of GXY triplets from human type III collagen around the collagenase cleavage site were inserted between two triple helix domains of the Scl2 bacterial collagen protein. The original bacterial CL domain was not cleaved by MMP-1 (collagenase 1) or MMP-13 (collagenase 3). The minimum type III sequence necessary for cleavage by the two collagenases was 5 GXY triplets, including 4 residues before and 11 residues after the cleavage site (P4-P11'). Cleavage of these chimeric substrates was not achieved by the catalytic domain of MMP-1 or MMP-13, nor by full-length MMP-3. Kinetic analysis of the chimeras indicated that the rate of cleavage by MMP-1 of the chimera containing six triplets (P7-P11') of collagen III was similar to that of native collagen III. The collagenase-susceptible chimeras were cleaved very slowly by trypsin, a property also seen for native collagen III, supporting a local structural relaxation of the triple helix near the collagenase cleavage site. The recombinant bacterial-human collagen system characterized here is a good model to investigate the specificity and mechanism of action of collagenases.     

Temperature-induced post-translational over-modification of type I procollagen. Effects of over-modification of the protein on the rate of cleavage by procollagen N-proteinase and on self-assembly of collagen into fibrils.

Previous observations suggested that incubating fibroblasts at elevated temperature caused over-modification of type I procollagen by post-translational enzymes because of a delay in folding of the collagen triple helix. Here, human skin fibroblasts were incubated at 40.5 instead of 37 degrees C, and the type I procollagen secreted into the medium was isolated. Analysis of the protein indicated that there was an increase of about 5 residues of hydroxylysine/alpha chain and about 1 residue of glycosylated hydroxylysine/alpha chain. Assays with procollagen N-proteinase indicated that the N-propeptide of the over-modified collagen was cleaved at a decreased rate, apparently because the over-modification altered the conformation-dependent cleavage site for the enzyme. Assays in a system for assembly of collagen into fibrils demonstrated that the over-modified protein had a higher critical concentration for self-assembly. Also, the fibrils formed from the over-modified collagen at 31 and 29 degrees C had smaller diameters than fibrils formed from normal type I collagen. The results provide direct evidence for earlier suggestions that post-translational over-modification of a fibrillar collagen can alter the morphology of the fibrils formed. The results also indicate that some of the biological consequences of the mutations in type I procollagen causing heritable disorders must be ascribed to the effects of post-translational over-modifications that frequently occur as secondary consequences of changes in the primary structure of the protein.     

Identification of the (183)RWTNNFREY(191) region as a critical segment of matrix metalloproteinase 1 for the expression of collagenolytic activity

Matrix metalloproteinase 1 (MMP-1) cleaves types I, II, and III collagen triple helices into (3/4) and (1/4) fragments. To understand the structural elements responsible for this activity, various lengths of MMP-1 segments have been introduced into MMP-3 (stromelysin 1) starting from the C-terminal end. MMP-3/MMP-1 chimeras and variants were overexpressed in Escherichia coli, folded from inclusion bodies, and isolated as zymogens. After activation, recombinant chimeras were tested for their ability to digest triple helical type I collagen at 25 degrees C. The results indicate that the nine residues (183)RWTNNFREY(191) located between the fifth beta-strand and the second alpha-helix in the catalytic domain of MMP-1 are critical for the expression of collagenolytic activity. Mutation of Tyr(191) of MMP-1 to Thr, the corresponding residue in MMP-3, reduced collagenolytic activity about 5-fold. Replacement of the nine residues with those of the MMP-3 sequence further decreased the activity 2-fold. Those variants exhibited significant changes in substrate specificity and activity against gelatin and synthetic substrates, further supporting the notion that this region plays a critical role in the expression of collagenolytic activity. However, introduction of this sequence into MMP-3 or a chimera consisting of the catalytic domain of MMP-3 with the hinge region and the C-terminal hemopexin domain of MMP-1 did not express any collagenolytic activity. It is therefore concluded that RWTNNFREY, together with the C-terminal hemopexin domain, is essential for collagenolytic activity but that additional structural elements in the catalytic domain are also required. These elements probably act in a concerted manner to cleave the collagen triple helix.     

Heterozygosity for a large deletion in the alpha 2(I) collagen gene has a dramatic effect on type I collagen secretion and produces perinatal lethal osteogenesis imperfecta

We characterized a de novo 4.5 kilobase pair deletion in the paternally derived alpha 2(I) collagen allele (COL1A2) from a patient with perinatal lethal osteogenesis imperfecta. The intron-to-intron deletion removed the seven exons which encode residues 586-765 of the triple helical domain of the chain. Type I procollagen molecules that contain the mutant pro-alpha 2(I) chain have a lower than normal thermal stability, undergo increased post-translational modification amino-terminal to the deletion junction, and are retained within the rough endoplasmic reticulum. The block to secretion appears to result from improper assembly of the triple helix, apparently a consequence of a disruption of charge-charge interactions between the shortened pro-alpha 2(I) chain and normal pro-alpha 1(I) chains. The lethal effect may be due to decreased secretion of normal collagen and secretion of a small amount of abnormal collagen that disrupts matrix formation.