Collagen Polypeptide Research Articles

Wet-spinnability and crosslinked fibre properties of two collagen polypeptides with varied molecular weight

The formation of naturally derived materials with wet stable fibrous architectures is paramount in order to mimic the features of tissues at the molecular and microscopic scale. Here, we investigated the formation of wet-spun fibres based on collagen-derived polypeptides with comparable chemical composition and varied molecular weight. Gelatin and hydrolysed fish collagen (HFC) were selected as widely available linear amino-acidic chains of high and low molecular weight, respectively, and functionalised in the wet-spun fibre state in order to preserve the material geometry in physiological conditions. Wet-spun fibre diameter and morphology were dramatically affected depending on the polypeptide molecular weight, wet-spinning solvent (i.e. 2,2,2-trifluoroethanol and dimethyl sulfoxide) and coagulating medium (i.e. acetone and ethanol), resulting in either bulky or porous internal geometry. Dry-state tensile moduli were significantly enhanced in gelatin and HFC samples following covalent crosslinking with activated 1,3-phenylenediacetic acid (Ph) (E: 726±43–844±85MPa), compared to samples crosslinked via intramolecular carbodiimide-mediated condensation reaction (E: 588±38MPa). Resulting fibres displayed a dry diameter in the range of 238±18–355±28μm and proved to be mechanically stable (E: 230kPa) following equilibration with PBS, whilst a nearly complete degradation was observed after 5-day incubation in physiological conditions.

Foam Cell Formation In Vivo Converts Macrophages to a Pro-Fibrotic Phenotype.

Formation of foam cell macrophages, which sequester extracellular modified lipids, is a key event in atherosclerosis. How lipid loading affects macrophage phenotype is controversial, with evidence suggesting either pro- or anti-inflammatory consequences. To investigate this further, we compared the transcriptomes of foamy and non-foamy macrophages that accumulate in the subcutaneous granulomas of fed-fat ApoE null mice and normal chow fed wild-type mice in vivo. Consistent with previous studies, LXR/RXR pathway genes were significantly over-represented among the genes up-regulated in foam cell macrophages. Unexpectedly, the hepatic fibrosis pathway, associated with platelet derived growth factor and transforming growth factor-β action, was also over-represented. Several collagen polypeptides and proteoglycan core proteins as well as connective tissue growth factor and fibrosis-related FOS and JUN transcription factors were up-regulated in foam cell macrophages. Increased expression of several of these genes was confirmed at the protein level in foam cell macrophages from subcutaneous granulomas and in atherosclerotic plaques. Moreover, phosphorylation and nuclear translocation of SMAD2, which is downstream of several transforming growth factor-β family members, was also detected in foam cell macrophages. We conclude that foam cell formation in vivo leads to a pro-fibrotic macrophage phenotype, which could contribute to plaque stability, especially in early lesions that have few vascular smooth muscle cells.

Pichia pastoris production of a prolyl 4-hydroxylase derived from Chondrosia reniformis sponge: A new biotechnological tool for the recombinant production of marine collagen

Prolyl 4-hydroxylase (P4H) is a α2β2 tetramer catalyzing the post-translational hydroxylation of prolines in collagen. Its recombinant production is mainly pursued to realize biotechnological tools able to generate animal contaminant-free hydroxylated collagen. One promising candidate for biomedical applications is the collagen extracted from the marine sponge Chondrosia reniformis, because of its biocompatibility and because is devoid of the health risks associated with bovine and porcine collagens.Here we report on the production and selection, by enzymatic and biomolecular analyses, of a triple transformed Pichia pastoris strain expressing a stable P4H tetramer derived from C. reniformis sponge and a hydroxylated non fibrillar procollagen polypeptide from the same animal. The percentage of recombinant procollagen hydroxylated prolines inside the transformed yeast was of 36.3% analyzed by mass spectrometry indicating that the recombinant enzyme is active on its natural substrate inside the yeast cell host. Furthermore, the recombinant sponge P4H has the ability to hydroxylate its natural substrate in both X and Y positions in the Xaa-Yaa-Gly collagenous triplets.In conclusion this Pichia system seems ideal for high-level production of hydroxylated sponge- or marine-derived collagen polypeptides as well as of conotoxins or other marine proteins of high pharmacological interest needing this particular post-translational modification.

Purification, characterization and antioxidant properties of low molecular weight collagenous polypeptide (37 kDa) prepared from whale shark cartilage (Rhincodon typus).

A low molecular weight type-II collagenous polypeptide (CIIp) from whale shark (WS) cartilage was prepared by thermolysin digestion; and examined for their physico-functional and antioxidant properties. The purified collagen was composed of an identical (α1)3 chains and was characterized as type-II. After hydrolysis with thermolysin, the α-chain of the WS collagen was degraded into smaller peptides with molecular weight ranging from 70 to 20KDa. CIIp was successfully separated from the hydrolysates with molecular weight of approximately 37kDa. Amino acid analysis of CII, and CIIp indicated imino acid contents of 155 and 121 amino acid residues per 1000 residues, respectively. Differing Fourier transform infrared (FTIR) spectra of CII and CIIp were observed, which suggested that the hydrolysis process by thermolysin affected the secondary structure and molecular order of collagen, particularly the triple-helical structure. The denaturation temperature of CII (34°C) was higher than that of CIIp. Low content of glycoprotein was observed in CII than CIIp due to removal of some polypeptides by thermolysin digestion. The antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl radicals and the reducing power of CIIp was greater than that of CII. The results proposed that the purified CIIp from WS cartilage with excellent antioxidant activities could be the suitable biomaterial for therapeutic applications.

Role of LARP6 and nonmuscle myosin in partitioning of collagen mRNAs to the ER membrane.

Type I collagen is extracellular matrix protein composed of two α1(I) and one α2(I) polypeptides that fold into triple helix. Collagen polypeptides are translated in coordination to synchronize the rate of triple helix folding to the rate of posttranslational modifications of individual polypeptides. This is especially important in conditions of high collagen production, like fibrosis. It has been assumed that collagen mRNAs are targeted to the membrane of the endoplasmic reticulum (ER) after translation of the signal peptide and by signal peptide recognition particle (SRP). Here we show that collagen mRNAs associate with the ER membrane even when translation is inhibited. Knock down of LARP6, an RNA binding protein which binds 5′ stem-loop of collagen mRNAs, releases a small amount of collagen mRNAs from the membrane. Depolimerization of nonmuscle myosin filaments has a similar, but stronger effect. In the absence of LARP6 or nonmuscle myosin filaments collagen polypeptides become hypermodified, are poorly secreted and accumulate in the cytosol. This indicates lack of coordination of their synthesis and retro-translocation due to hypermodifications and misfolding. Depolimerization of nonmuscle myosin does not alter the secretory pathway through ER and Golgi, suggesting that the role of nonmuscle myosin is primarily to partition collagen mRNAs to the ER membrane. We postulate that collagen mRNAs directly partition to the ER membrane prior to synthesis of the signal peptide and that LARP6 and nonmuscle myosin filaments mediate this process. This allows coordinated initiation of translation on the membrane bound collagen α1(I) and α2(I) mRNAs, a necessary step for proper synthesis of type I collagen.

Research on Preparation Process of Collagen Polypeptide from Pig Skin by Two‐Step‐Hydrolysis (LB397)

In this study, neutral proteinase, alkaline proteinase, and papain were used to hydrolyze fresh pig skin respectively. The effects of pH value ,temperature, enzyme content and substrate content on hydrolysis were studied by the single factor test, and orthogonal design, by assessing the parameter ‐ degree of hydrolysis (DH), was adopted to determine the optimum condition of hydrolysis. The optimal hydrolysis condition of neutral proteinase was: pH 6.5, 45°C, enzymatic content 0.5%, and substrate content 15%; while The optimal hydrolysis condition of alkaline proteinase was: pH 11, 55°C, enzymatic content 0.5%, and substrate content 35%; and the most suitable hydrolysis parameters of papain were: pH 5, temperature of 60°C, 0.9% of enzyme content and 30% of substrate content. The ability of hydrolysis was sequenced as: alkaline proteinase > papain > neutral proteinase. On the basis of the optimum condition of each single proteinase, any two of the proteinases were chosen to hydrolyze fresh pig skin by two‐step‐hydrolysis. The results showed that any combination of the enzymes has a higher DH value than its single component and the adding sequence inflects DH value. Overall, the two‐step‐hydrolysis of papain plus alkaline shows the highest DH value (22.31%).Grant Funding Source: Supported by Science and Technology Department of Hebei Province, project no. 14227114D

Extraction Optimization and Characterization of Collagen from the Lung of Soft-Shelled Turtle Pelodiscus Sinensis

The soft-shelled turtle (Pelodiscus sinensis) is a commercially important aquatic species with abundant collagen content and precious nutritious high quality goods. The present study was to optimize the collagen extraction from lung of Pelodiscus sinensis. Single-factor test was employed to investigate the effects of different extraction methods and the major factors that influence the collagen production in enzymatic extraction method on the collagen yield of lung. Optimization of the papain enzymatic extraction parameters was then examined using an orthogonal test design L9 (34). The optimum extraction conditions were obtained when the extraction temperature, papain enzyme dose, the ratio of solid to solution, and extraction time were 32°C, 4.0mg/ml, 1:35, and 12h, respectively. Under the optimized conditions, the collagen yield was up to 79.29%. The properties of turtle lung collagen were characterized by SDS-PAGE, UV scanning, and amino acid analysis. The results showed that the extracted lung collagen had high imino acid content at 21.8% and contained two α chains, β, and γ-components, belonging to the typical type I collagen. The amphibious aquatic turtle collagen is thought to be a viable novel source for replacement of terrestrial mammals and could provide scientific reference for the development of collagen polypeptide for terrapin animals.

Tacrolimus (FK506) prevents early stages of ethanol induced hepatic fibrosis by targeting LARP6 dependent mechanism of collagen synthesis.

Tacrolimus (FK506) is a widely used immunosuppressive drug. Its effects on hepatic fibrosis have been controversial and attributed to immunosuppression. We show that in vitro FK506, inhibited synthesis of type I collagen polypeptides, without affecting expression of collagen mRNAs. In vivo, administration of FK506 at a dose of 4 mg/kg completely prevented development of alcohol/carbon tetrachloride induced liver fibrosis in rats. Activation of hepatic stellate cells (HSCs) was absent in the FK506 treated livers and expression of collagen α2(I) mRNA was at normal levels. Collagen α1(I) mRNA was increased in the FK506 treated livers, but this mRNA was not translated into α1(I) polypeptide. No significant inflammation was associated with the fibrosis model used. FK506 binding protein 3 (FKBP3) is one of cellular proteins which binds FK506 with high affinity. We discovered that FKBP3 interacts with LARP6 and LARP6 is the major regulator of translation and stability of collagen mRNAs. In the presence of FK506 the interaction between FKBP3 and LARP6 is weakened and so is the pull down of collagen mRNAs with FKBP3. We postulate that FK506 inactivates FKBP3 and that lack of interaction of LARP6 and FKBP3 results in aberrant translation of collagen mRNAs and prevention of fibrosis. This is the first report of such activity of FK506 and may renew the interest in using this drug to alleviate hepatic fibrosis.

RNA protein interactions governing expression of the most abundant protein in human body, type I collagen

Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half-life of collagen messenger RNAs (mRNAs) and their increased translatability. Type I collagen is composed of two α1 and one α2 polypeptides that fold into a triple helix. This stoichiometry is strictly regulated to prevent detrimental synthesis of α1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen α1(I) and α2(I) polypeptides. Collagen α1(I) mRNA has in the 3' untranslated region (UTR) a C-rich sequence that binds protein αCP, this binding stabilizes the mRNA in collagen producing cells. In the 5' UTR both collagen mRNAs have a conserved stem-loop (5' SL) structure. The 5' SL is critical for high collagen expression, knock in mice with disruption of the 5' SL are resistant to liver fibrosis. the 5' SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of α1(I) and α2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only α1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments.

Association of Calcium and Phosphate Ions with Collagen in the Mineralization of Vertebrate Tissues

Among the vertebrate species, collagen is the most abundant protein and is associated with mineralization of their skeleton and dentition in all tissues except enamel. In such tissues, bones, calcifying tendon, dentin, and cementum are comprised principally of type I collagen, which has been proposed as a template for apatite mineral formation. Recent considerations of the interaction between type I collagen and calcium and phosphate ions as the major constituents of apatite have suggested that collagen polypeptide stereochemistry underlies binding of these ions at sites within collagen hole and overlap regions and leads to nucleation of crystals. The concept is fundamental to understanding both normal and abnormal mineralization, and it is reviewed in this article. Given this background, avenues for additional research studies in vertebrate mineralization will also be described. The latter include, for instance, how mineralization events subsequent to nucleation, that is, crystal growth and development, occur and whether they, too, are directed by collagen stereochemical parameters; whether mineralization can be expected in all spaces between collagen molecules; whether the side chains of charged amino acid residues actually point toward and into the hole and overlap collagen spaces to provide putative binding sites for calcium and phosphate ions; and what phenomena may be responsible for mineralization beyond hole and overlap zones and into extracellular tissue regions between collagen structural units. These questions will be discussed to provide a broader understanding of collagen contributions to potential mechanisms of vertebrate mineralization.

Prolyl 4-Hydroxlase Activity Is Essential for Development and Cuticle Formation in the Human Infective Parasitic Nematode Brugia malayi

Collagen prolyl 4-hydroxylases (C-P4H) are required for formation of extracellular matrices in higher eukaryotes. These enzymes convert proline residues within the repeat regions of collagen polypeptides to 4-hydroxyproline, a modification essential for the stability of the final triple helix. C-P4H are most often oligomeric complexes, with enzymatic activity contributed by the α subunits, and the β subunits formed by protein disulfide isomerase (PDI). Here, we characterize this enzyme class in the important human parasitic nematode Brugia malayi. All potential C-P4H subunits were identified by detailed bioinformatic analysis of sequence databases, function was investigated both by RNAi in the parasite and heterologous expression in Caenorhabditis elegans, whereas biochemical activity and complex formation were examined via co-expression in insect cells. Simultaneous RNAi of two B. malayi C-P4H α subunit-like genes resulted in a striking, highly penetrant body morphology phenotype in parasite larvae. This was replicated by single RNAi of a B. malayi C-P4H β subunit-like PDI. Surprisingly, however, the B. malayi proteins were not capable of rescuing a C. elegans α subunit mutant, whereas the human enzymes could. In contrast, the B. malayi PDI did functionally complement the lethal phenotype of a C. elegans β subunit mutant. Comparison of recombinant and parasite derived material indicates that enzymatic activity may be dependent on a non-reducible covalent link, present only in the parasite. We therefore demonstrate that C-P4H activity is essential for development of B. malayi and uncover a novel parasite-specific feature of these collagen biosynthetic enzymes that may be exploited in future parasite control.

Stimuli responsive deswelling of radiation synthesized collagen hydrogel in simulated physiological environment

Collagen hydrogels were prepared via radiation crosslinking. The simulated physiological environmental effects related to their biomedical applications on the volume phase transition of collagen hydrogel were studied, that is stimuli response to ions, temperature, and pH. The deswelling behavior of collagen hydrogel depends on the salt concentration, temperature, pH, and the hydrogel preparation procedure. Meanwhile, hydrogel structure related to the volume phase transition was investigated by FTIR, fluorescence spectrum, and HR-MAS NMR. Deswelling in salt solution caused little change on collagen conformation, and a denser network led to more significant tyrosine-derived fluorescence quenching. Hydrogen bonding between hydrated water and collagen polypeptide chain was dissociated and the activity of hydrophobic side chain increased, inducing a higher extent of contraction with the increasing of salt concentration. Moreover, salt solution treatments weakened the electrostatic interactions, side chains interactions, and hydrogen bonding of collagen hydrogel, which reduced the thermal stability of collagen hydrogel. Comparing with cell-free collagen hydrogel contraction, fibroblasts did not aggravate contraction of collagen hydrogel significantly. This study elucidated the deswelling mechanism of radiation crosslinked collagen hydrogel in simulated physiological environment and provides strategies for controlling the stimuli response of collagen hydrogel in biomedical application.

UVB irradiation down‐regulates type XVI collagen expression in mouse and human skin

Type XVI collagen is a member of the fibril-associated collagens with interrupted triple helices; however, its function or regulation remain unclear. This study is to examine the effect of ultraviolet B (UVB) or photoaging on type XVI collagen expression in various cultured cells, mouse, and human skin. The level of α1 (XVI) collagen mRNA was determined by quantitative real-time reverse transcriptase-polymerase chain reaction and the localization of type XVI collagen in normal human skins was detected by theα1 (XVI) collagen polypeptide antibody. Exposure of keratinocytes resulted in suppression of mRNA level in a dose- and time-dependent manner and in normal fibroblasts or organotypic cocultures was also inhibited. Expression level in hairless mouse skin was decreased by UVB exposure. Messenger RNA level of human skins in the sun-protected area appeared to be greater than that in the sun-exposed area. Sun-protected and sun-exposed normal skin taken from young subjects showed positive immunoreactivities with the anti-α1 (XVI) collagen antibody in the subepidermal region, whereas sun-exposed skin from elderly subjects exhibited negative immunoreaction. Reduction of type XVI collagen by UVB irradiation in vitro and in vivo may be related to the alteration of extracellular matrix in the photodamaged skin.

Moisture absorption and retention properties, and activity in alleviating skin photodamage of collagen polypeptide from marine fish skin

Collagen polypeptides were prepared from cod skin. Moisture absorption and retention properties of collagen polypeptides were determined at different relative humidities. In addition, the protective effects of collagen polypeptide against UV-induced damage to mouse skin were evaluated. Collagen polypeptides had good moisture absorption and retention properties and could alleviate the damage induced by UV radiation. The action mechanisms of collagen polypeptide mainly involved enhancing immunity, reducing the loss of moisture and lipid, promoting anti-oxidative properties, inhibiting the increase of glycosaminoglycans, repairing the endogenous collagen and elastin protein fibres, and maintaining the ratio of type III to type I collagen.

Preparation and characterization of collagen polypeptide chelated calcium from fish bone powder of channel catfish(Ictalurus punctatus)

Calcium is essential for living organisms.Even with an apparently sufficient intake of dietary calcium,there is some concern that inadequate calcium is absorbed by the small intestine,due to precipitation of insoluble calcium salts in the neutral to slightly basic intestinal lumen.Some studies revealed that peptides have the capacity to chelate Ca and to prevent the precipitation of insoluble calcium salts,thereby increasing the amount of soluble Ca availability for absorption across the mucosa.However,the mechanism and degree of calcium ion binding are still unclear.In this paper,the combinations between collagen polypeptide(from the enzymatic hydrolysate of channel catfish bone powder)and calcium ion were studied by measuring chelate rate.On the basis of results of single-factor experiments,the Box-Behnken central composite design and response surface method were adopted to obtain the optimum conditions for chelation.The optimal chelate conditions were determined as:chelate temperature 60 ℃,chelate pH 5.4,chelate time 1.5 h and ratio of collagen polypeptide to calcium 2∶ 1(W/W).Under the optimized conditions,the chelate rate of Ca-collagen polypeptide could reach 82.53%.The formation of Ca-collagen polypeptide chelate was confirmed by the UV-VIS and FT-IR spectra.The characterization of amino acid composition of the chelate was similar to typical collagen-like protein.This research provides a practical guideline and a theoretical basis for fully utilizing fish bone protein resources and developing the deep processed products.

Preparation and Antioxidant Properties of Collagen Polypeptide from <i>Navodon septentionalis</i> Bone

Collagen peptide was prepared from Navodon septentionalis bone by applying bromelin as a catalyst. Navodon septentionalis bone was mixed with 1%bromelin (w:w)and water at a ratio of 1:4 (w/w).The mixture was incubated at 60°Cfor 4 hours. The dehydration degree was up to 26.20% on optimum condition and the concentration of polypeptide was almost 50mg/ml. The antioxidant activity was investigated and compared with vitamin C, TBHQ, BHA and BHT. Collagen polypeptide at 50mg/ml and 10mg/ml exhibited 80% and 92.5% of scavenging activities on hydroxy radical and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, respectively.

A novel homozygous splice site mutation in COL7A1 in a Chinese patient with severe recessive dystrophic epidermolysis bullosa and squamous cell carcinoma

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils in the dermo-epidermal junction. The development of cutaneous squamous cell carcinoma (SCC) is one of the most serious complications of this disease. We report herein a Chinese patient with the severe generalized subtype of RDEB (RDEB-sev gen) complicated by SCC. Skin biopsies were examined for histology, basement membrane ultrastructure, and type VII collagen expression. Genomic DNA was extracted from the peripheral blood samples and subjected to polymerase chain reaction amplification and direct automated DNA sequencing. Histopathological examination of the patient's skin revealed an undetectable expression of type VII collagen polypeptides in the basement membrane zone. Mutation analysis identified a novel splice site mutation in intron 64 (IVS64+5g->a) of COL7A1 gene, which resulted in an in-frame deletion of exon 64 in both alleles. This report contributes to the expanding database of COL7A1 mutations and emphasizes the need to elucidate the underlying genetic mechanisms associated with the increased incidence of SCC in RDEB patients.

Revertant Mosaicism Due to a Second-Site Mutation in COL7A1 in a Patient with Recessive Dystrophic Epidermolysis Bullosa

Despite the high incidence of revertant mosaicism (35%) in patients with the genetic skin disease epidermolysis bullosa (EB) due to correcting mutations in the genes COL17A1 and LAMB3, revertant mosaicism has not been described for COL7A1 until recently. Mutations in COL7A1 are responsible for the most devastating form of EB in adults, which is characterized by cocooned "mitten" deformities of the hands. This report shows in vivo reversion of an inherited COL7A1 mutation in a patient with recessive dystrophic EB who was homozygous for the frameshift mutation COL7A1:c.6527insC,p.2176FsX337. The patient exhibited a patch of clinically healthy revertant skin on her left forearm. The second-site mutation c.6528delT, which is present in revertant keratinocytes, resulted in correction of the reading frame. As the new CCC codon codes for the same amino acid proline as the wild-type codon CCT, the revertant cells expressed wild-type type VII collagen polypeptide, leading to restoration of skin function. We hypothesize that, on careful examination, revertant mosaicism might be found to be more common in patients with type VII collagen-deficient EB. Furthermore, the revertant keratinocytes might offer the possibility to explore cell-based therapeutic strategies, by culturing in vitro and subsequently grafting as part of bioengineered dermo-epidermal substitutes on affected skin.

Prolyl hydroxylase in human corpora lutea during menstrual cycle and early pregnancy

Immunohistochemical staining for human prolyl hydroxylase revealed intense staining of the human corpora lutea (CL) parencyma during early pregnancy compared with those in the menstrual cycle. These results suggest that human prolyl hydroxylase might play an important role in determining the physiology and structure of the CL during the menstrual cycle and early pregnancy.

Binding of LARP6 to the Conserved 5′ Stem–Loop Regulates Translation of mRNAs Encoding Type I Collagen

Type I collagen is the most abundant protein in the human body, produced by folding of two α1(I) polypeptides and one α2(I) polypeptide into the triple helix. A conserved stem–loop structure is found in the 5′ untranslated region of collagen mRNAs, encompassing the translation start codon. We cloned La ribonucleoprotein domain family member 6 (LARP6) as the protein that binds the collagen 5′ stem–loop in a sequence-specific manner. LARP6 has a distinctive bipartite RNA binding domain not found in other members of the La superfamily. LARP6 interacts with the two single-stranded regions of the 5′ stem–loop. The K d for binding of LARP6 to the 5′ stem–loop is 1.4 nM. LARP6 binds the 5′ stem–loop in both the nucleus and the cytoplasm. In the cytoplasm, LARP6 does not associate with polysomes; however, overexpression of LARP6 blocks ribosomal loading on collagen mRNAs. Knocking down LARP6 by small interfering RNA also decreased polysomal loading of collagen mRNAs, suggesting that it regulates translation. Collagen protein is synthesized at discrete regions of the endoplasmic reticulum. Using collagen–GFP (green fluorescent protein) reporter protein, we could reproduce this focal pattern of synthesis, but only when the reporter was encoded by mRNA with the 5′ stem–loop and in the presence of LARP6. When the reporter was encoded by mRNA without the 5′ stem–loop, or in the absence of LARP6, it accumulated diffusely throughout the endoplasmic reticulum. This indicates that LARP6 activity is needed for focal synthesis of collagen polypeptides. We postulate that the LARP6-dependent mechanism increases local concentration of collagen polypeptides for more efficient folding of the collagen heterotrimer.