View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector 022-202X/82/7903-003s$02.00/0 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, 79:38-68, 1982 Vol. 79, Supplement I Copyright © 1982 by The Williams & Wilkins Co. Printed in U.S.A. REPORTS How Does a Skin Fibroblast Make Type I Collagen Fibers? DARWIN J. PROCKOP, M.D., PH.D. Department of Biochemistry, College of Medicine and Dentistry, of New Jersey-Rutgers Medical School, Piscataway, New Jersey, U.S.A. We now have an impressive amount of information proa2(I) chain [9]. By using a :l2P-Iabeled cloned cDNA and the about several steps in the synthesis of type I collagen technique known as "Southern blotting," it was Rhown that fibers by skin fibroblasts: (a) the transcription of the DNA fragments arising from the human proa2(I) gene were genes for proal(I) and proa2(I) polypeptide chains; (b) obtained only from mouse-human cell hybrids and hamster­ the processing of the initial RNA transcripts of the genes human cell hybrids which contained the human chromosome to mature mRNAs; (c) translation of the mRNAs to pre­ #7. proal(I) and preproa2(I) chains; (d) processing of the In the second step, the genes for proal (I) and proa2(I) chains preproal(I) and preproa2(I) chains to procollagen; and are transcribed into RNA. Experiments carried out first with a (e) the further processing of procollagen to collagen proa2(I) gene from chick embryos [10-12], then with a proa2(I) fibers. The speed with which we are currently develop­ gene from sheep [13] and finally a pro(�2(l) gene from man ing this information about collagen and comparable in­ [14,15], demonstrated that both the gene and the initial RNA formation about other constituents of skin suggests that transcript of the collagen gene are about 40,000 bases, or eight skin may well be the first human organ which we fully times longer than the nucleotides necessary to code for the understand. protein. The large size of the gene and the initial RNA transcript is accounted for by the presence of about 50 separate interven­ ing sequences (Fig 1). Most of the intervening sequences divide As the initial speaker at this conference, I thought it might the central portion of the gene, the part which codes for the a be helpful to set the stage by reviewing the information we now chain domain, into short coding sequences or exons of 54 or 108 have about the question, "How does a fibroblast make type I bases. The 54 or 108 base exons code for 18 or 36 amino acid collagen, the most abundant constituent of skin?" (Table). sequences with the structure (Gly-X-Y)6 or (Gly-X-Yh2 (10-12). The synthesis of any extracellular protein occurs in at least Occasionally the exons are 49 and 95 bases [12]. These obser­ 6 steps: (a) The structural gene for the protein is "turned on" vations have led to the suggestion that the collagen gene may in the sense of being prepared for transcription; (b) the DNA of have originated by extensive duplication of a primitive collagen the structural gene is transcribed into an initial RNA transcript; of 54 bases [10-12]. Alternatively, it is possible that the large (c) the initial RNA transcript is processed by a series of modi­ number of intervening sequences arose during evolution to fications to a mature mRNA; (d) the mRNA is translated into prevent recombination events (crossover mutations), which fre­ a polypeptide chain; (e) the polypeptide chain is processed by quently occur in highly repetitive DNA sequences. one or more modifying enzymes; (f) the polypeptide chain folds In the third step of collagen synthesis, the hnRNAs are into the correct three-dimensional conformation and is further processed to functional mRNAs. Because there are 50 interven­ moditied so that its biological activity is expressed. ing sequences, over 50 enzymic steps are required. In fact, In considering the synthesis of type I collagen (for more examination of one part of this process has suggested that the detailed reviews, see references 1-7), we know relatively little excision of a single large intervening sequence may occur in about the first step, the "turning on" of the structural genes for more than one step [16]. The RNA must also be processed proal (I) and proa2(l) polypeptide chains which comprise the through steps that add a "cap" containing 7-methylguanosine molecule. In fact, although we have considerable information to its 5' -end, and a stretch of 20-100 adenine nucleotides to the about regulation of the operon and similar gene systems in lac :1'-end, much as occurs with most other messengers of eukar­ E. coli, we have almost no information about the regulatory yotic cells. Recent observations on several types of protein mechanisms which turn on genes in eukaryotic cells. What we mRNAs have indicated that the length of the untranslated know about collagen genes is that most cells, particularly cells nucleotides at the 3' -end of the mRNAs for a given protein may in culture, either synthesize collagen or contain some of the vary [17]. Therefore, transcription of a single gene can give rise necessary components for collagen synthesis such as active to 2 or more different length mRNAs coding for the same prolylhydroxylase [8]. We also know that the gene system for protein. In the case of type I pro collagen, mRNAs of several collagen synthesis is among the oldest gene systems in the different sizes have been identified for the proa2(I) chain [15]. multi-cellular organisms, and it dates as far back in evolution The significance of this variability in the size of the mRNAs as the origin of sponges [2]. Therefore, it seems that synthesis coding for a given protein is unclear. of extracellular collagen fibers is a major mechanism which In the fourth step, the mRNAs are translated into preproal(I) nature has developed for holding together cells to form a multi­ and preproa2(I) chains. As appears to be true of all protein cellular organism and is an intrinsic property of many cell types. synthesized for export from cells, the amino terminal ends of the We have one further point of information about collagen at the newly-synthesized polypeptides contain a stretch of hydropho­ level of regulation of the gene in that we know the chromosomal bic amino acids which direct binding of the ribosome to the location of the proa2(I) chain. The proa2(I) gene was assigned membranes of the rough endoplasmic reticulum. This region of to chromosome #7 by use of a cloned cDNA for the human the protein, called a "signal sequence" or "pre-sequence," is rapidly cleaved after the newly synthesized polypeptide chain This work was supported by in part by Grant AM-16516 from the National Institutes of Health. passes through the membrane of the rough endoplasmic retic­ Reprint requests to: Darwin J. Prockop, M.D., Ph.D., Department ulum into the cisternae of the organelle. In the case of collagen of Biochemistry, College of Medicine and Dentistry of New .Jersey, synthesis, both the proal(I) [18,19] and the proa2(I) [19] chains Rutgers Medical School, Piscataway, NJ 08854. have been shown to have signal peptides, and the signal pep- 3s 4s PROCKOP Vol. 79, Supplement 1 How does a skin fibroblast make type Collagen? In the sixth step, the secreted procollagen is converted into I ----- collagen fibers (Fig 3). Here at least 2 separate procollagen 1. Genes for prOa I (I) and proa2 (I) are "turned on." 2. DNA is transcribed into hnRNAs. proteinases are involved [20-25]. One, procollagen amino-pro­ -about 50 intervening sequences. teinase, removes the amino-propeptides from the 3 proa chains. -hnRNA about 8 times longer than coding sequences. Another procollagen carboxy-proteinase, removes the carboxy­ 3. hnRNAs are processed to mRNAs. propeptides. The amino- and carboxy-propeptides constitute -over 50 steps each for proal (I) and proa2 (I) about one-third of the procollagen molecule. With the removal -2 size mRNAs for proal (I); proa2 3 for (I) of the propeptides, the biological activity of the protein is 4. mRNAs are translated into preproal and preproa2 chains. (1) (I) released and the resulting collagen self-assembles into fibers 5. Preproal (I) and preproa2 (ll chains are processed to procoUagen. -over 11 enzymes. [2;3). Finally, in steps not discussed here, the collagen molecules -over 120 steps. which have self-assembled into fibers are cross-linked by a 6. Procollagen is converted to collagen fibers. series of enzymic and nonenzymatic steps so that large sections -at least 3 enzymes of the fiber become a single, covalently-linked structure. -one-third of the protein discarded At least 3 important generalizations emerge from this infor­ -the collagen self-assembles into fibers mation about the synthesis of type I collagen fibers by skin -the fibers are cross-linked. fibroblasts. One of these generalizations is that the structural and functional relationships of the collagen molecule are re­ markably simple. Each of the 3 a chains contain a large number DNA of repeating sequences of -Gly-Pro-Hyp-. These triplets in the polypeptide chains direct folding of the chains into the unique rope-like, triple-helical conformation which is characteristic of Transcription of 40,000 bose gene and addition of the collagen molecule. The remainder of the a chains consist of poly A 01 3' end. j triplets of -Gly-X-Y- in which the side chains of the amino acids in the X- and Y-positions point away from the center of the triple-helix and thereby provide precisely positioned reactive RNA -------------------------------------------- (A)n groups that direct lateral and longitudinal assembly of the molecules into fibers.